Ultraconserved Regions Encoding ncRNAs

ABSTRACT

Described herein are methods for differentiate human cancers comprising using one or more transcribed ultraconserved regions (T-UCR) expression profiles where the association between the genomic location of UCRs and the analyzed cancer-related genomic elements is highly statistically significant and comparable to that reported for miRNAs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/963,329 filed Aug. 3, 2007, and PCT/US2008/xxxxx filed xxxxx, 2008,the entire disclosure of which is expressly incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was not made with any Government support and theGovernment has no rights in this invention.

BACKGROUND

Taken as a whole, cancers are a significant source of mortality andmorbidity in the U.S. and throughout the world. However, cancers are alarge and varied class of diseases with diverse etiologies. Researcherstherefore have been unable to develop treatments or diagnostic testswhich cover more than a few types of cancer.

For example, cancers are associated with many different classes ofchromosomal features. One such class of chromosomal features areperturbations in the genomic structure of certain genes, such as thedeletion or mutation of tumor suppressor genes. The activation ofproto-oncogenes by gene amplification or promoter activation (e.g., byviral integration), epigenetic modifications (e.g., a change in DNAmethylation) and chromosomal translocations can also causecancerigenesis. Such perturbations in the genomic structure which areinvolved in the etiology of cancers are called “cancer-associatedgenomic regions” or “CAGRs.”

Chromosomal fragile sites are another class of chromosomal featureimplicated in the etiology of cancers. Chromosomal fragile sites areregions of genomic DNA which show an abnormally high occurrence of gapsor breaks when DNA synthesis is perturbed during metaphase. Thesefragile sites are categorized as “rare” or “common.” As their namesuggests, rare fragile sites are uncommon. Such sites are associatedwith di- or tri-nucleotide repeats, can be induced in metaphasechromosomes by folic acid deficiency, and segregate in a Mendelianmanner. An exemplary rare fragile site is the Fragile X site.

Common fragile sites are revealed when cells are grown in the presenceof aphidocolin or 5-azacytidine, which inhibit DNA polymerase. At leasteighty-nine common fragile sites have been identified, and at least onesuch site is found on every human chromosome. Thus, while their functionis poorly understood, common fragile sites represent a basic componentof the human chromosome structure.

Induction of fragile sites in vitro leads to increased sister-chromatidexchange and a high rate of chromosomal deletions, amplifications andtranslocations, while fragile sites have been colocalized withchromosome breakpoints in vivo. Also, most common fragile sites studiedin tumor cells contain large, intra-locus deletions or translocations,and a number of tumors have been identified with deletions in multiplefragile sites. Chromosomal fragile sites are therefore mechanisticallyinvolved in producing many of the chromosomal lesions commonly seen incancer cells.

All malignant cells have specific alterations at DNA loci that encodegenes for oncoproteins or tumor suppressors (Balmain et al., 2003;Wooster and Weber, 2003). This common feature has recently been expandedto include a large class of non-coding RNAs (ncRNAs) called microRNAs(miRNAs) (Ambros, 2004) that are also involved in cancer initiation andprogression (Calin et al., 2002; Croce and Cann, 2005; Berezikov andPlasterk, 2005a; Esquela-Kerscher and Slack, 2006; Calin and Croce,2006a). mRNAs affect the regulation of gene expression at both thetranscriptional and post-transcriptional levels (Ambros, 2003; Ambros,2004).

The extent of involvement of miRNAs and the involvement of other classesof ncRNAs in human tumorigenesis is unknown. Therefore, there is a needfor further research into the molecular mechanisms and signaltransduction pathways altered in cancer.

There is a further need for the identification of new molecular markersand potential therapeutic agents.

The ultraconserved regions (UCRs) of the human genome (Bejerano et al.,2004b) are also miRNAs that are almost completely conserved amongvarious species (Berezikov et al., 2005b). For example, the activemolecules of the miR-16-1/miR-15a cluster, has been shown to be anessential player in the initiation of chronic lymphocytic leukemia (CLL)(Calin et al., 2005a), and are completely conserved in human, mouse andrat and highly conserved in nine out of the ten sequenced primatespecies (Berezikov et al., 2005b). Comparative sequence analysis hasidentified a number of highly conserved genomic sequences. Some of theseregions do not produce a transcript that is translated into protein andare therefore considered to be non-genic. Various names have beenapplied to this class of sequences: conserved non-genic sequences (CNGs)(Dermitzakis et al., 2005), conserved non-coding sequences (CNSs/CNCs)(Meisler, 2001), multiple species conserved sequences (MCSs) (Thomas etal., 2003) or highly conserved regions (HCRs) (Duret et al., 1993).

UCRs are a subset of conserved sequences that are located in both intra-and inter-genic regions. They are absolutely conserved (100%) betweenorthologous regions of the human, rat, and mouse genomes (Bejerano etal., 2004b). In contrast to other regions of conserved sequence, 53% ofthe UCRs have been classified as non-exonic (‘N’, 256/481 withoutevidence of encoding protein), while the other 47% have been designatedeither exonic (‘E’, 111/481, that overlap mRNA of known protein-codinggenes), or possibly exonic (‘P’, 114/481, with inconclusive evidence ofoverlap with protein coding genes).

A large portion of transcription products of the non-coding functionalgenomic regions have significant RNA secondary structures and arecomponents of clusters containing other sequences with functionalnon-coding significance (Bejerano et al., 2004a). The UCRs represent asmall fraction of the human genome that are likely to be functional butnot encoding proteins, and have been called the “dark matter” of thehuman genome (Bejerano et al., 2004a). Because of the high degree ofconservation, the UCRs may have fundamental functional importance forthe ontogeny and phylogeny of mammals and other vertebrates. This wasillustrated by the recent finding of a distal enhancer and anultraconserved exon derived from a novel retroposon active inlobe-finned fishes and terrestrial vertebrates more than 400 millionyears ago and maintained as active in a “living fossil” coelacanth(Bejerano et al., 2006).

Further experimental proof of the functional importance of UCRs is basedon analysis of mice with targeted mutations. Megabase deletions of genedeserts that lack ultraconserved elements or highly conserved sequencesresulted in viable mice that developed apparently without detectablephenotypes (Nobrega et al., 2004). By contrast, gene deserts containingseveral UCRs (such as the two gene deserts surrounding the DA CHI geneon human chromosome 13g21.33) were shown to contain long-rangeenhancers, some of them composed of UCR sequences (Nobrega et al.,2003).

In spite of considerable research into therapies for cancer-relateddiseases, these diseases remain difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for diagnosing and/or treating cancer. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF INVENTION

Described herein is a thorough genomic interrogation of the status ofUCRs in a large panel of human leukemias and carcinomas.

We investigated the genome-wide expression of UCRs in various normal andcancer samples, and we assessed the relationship between the genomiclocation of these sequences and the known regions involved in cancers.

Furthermore, we identified a functional role for miRNAs in thetranscriptional regulation of cancer-associated UCRs.

Also described herein is evidence in cancer systems that adifferentially expressed UCR could alter the functional characteristicsof malignant cells.

Also described herein, by combining these data with the elaborate modelsinvolving miRNAs in human tumorigenesis, is a model in which alterationin both coding and non-coding RNAs cooperate in the initiation andprogression of malignancy.

In one broad aspect, there is described herein are methods fordifferentiate human cancers comprising using one or more transcribedultraconserved regions (T-UCR) expression profiles where the associationbetween the genomic location of UCRs and the analyzed cancer-relatedgenomic elements is highly statistically significant and comparable tothat reported for miRNA

—Insert Summary of Claims Here—

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1E. Transcriptional characteristics of various types of UCRs:

FIG. 1A. Northern blots showing the expression of various UCRs in normaltissues. In the case of uc.246(E) and uc.269A(N), the presence of thelong transcript was confirmed by the RACE cloning experiments. For sometissues, duplicate samples were procured to confirm reproducibility.Normalization was performed with U6. The arrows on the left side showthe identified transcripts.

FIG. 1B. T-UCRs 291 and 73A expression (normalized to 18S rRNA) wasconfirmed by qRT-PCR (graphs) and microarray analyses (Normalized numberunder the graph) in normal CD5+/CD19+ lymphocytes and malignant CLLsamples. P-values were significant for both qRT-PCR and microarray datastatistical comparison. Each box represents the distribution ofexpression measured for normals (blue) and CLL patients (red), ends ofthe boxes define the 25th and 75th percentiles, a line indicates themedian, bars define the 10th and 90th percentiles.

FIG. 1C. Number of UCRs expressed in one or more of 19 tissues, asrevealed by microarray analysis; UCR type (E, N, P) numbers areindicated. Four types of transcription were found: ubiquitouslyexpressed UCRs (in 18 or 19 out of 19 different tissues), UCRs expressedin the majority of tissues (10 to 17), UCRs expressed in a minority oftissues (2 to 9) and tissue-specifically expressed UCRs.

FIG. 1D. Percentage of each UCR type (E, N, P) that is ubiquitouslytranscribed (both uni- and bi-directionally) in all the analyzedtissues; the absolute numbers for each UCR type are shown in the boxes.

FIG. 1E. Expression of the sense or antisense strand UCRs 73, 133 and269, relative to 18S rRNA, in CD19+ B cells from three different donors.Sense/antisense strand specific real-time RT-PCR was used to validatethe strand specific expression of the UCRs observed with microarrayanalysis; the average+1-standard deviation of microarray results forCDS+samples is under each graph. Microarray probes are named as follows:the sense genomic probe is named “+”, while the probe to thecomplementary sequence is named “A+”.

FIGS. 2A-2B. Hierarchical clustering of tissues and tumors according toUCRs expression. Unsupervised cluster of (FIG. 2A) 22 normal humantissues and (FIG. 2A\B) 133 leukemias and carcinomas made using thenon-exonic UCRs of the chip. Some of the T-UCRs that well differentiatethe tissue types (FIG. 2A) or carcinomas from leukemias (FIG. 2B) areexpanded at the right. Samples are in columns, T-UCRs in rows. A greencolored gene is down-regulated compared to its median expression in allsamples, red is up-regulated and yellow means no variation. The completeUCRs profile of tissues and tumors can be found in FIGS. 5 and 6.

FIGS. 3A-3E. T-UCRs represent direct targets of miRNAs:

FIG. 3A. Examples of sites of complementarity T-UCR::miRNA. Theuc.348::miR-155 pairing is shown as an example of low levels ofcomplementarity in contrast with the other 4 interacting paired genesfor which higher levels of complementarity are found.

FIG. 3B. The correlation by qRT-PCR for miR-155, uc.160 and uc.346Aexpression in 9 CLL patients. Lymphocytes from four differentindividuals were used as normal controls.

FIG. 3C. The direct miRNA::T-UCR interaction. Relative repression offirefly luciferase expression standardized to a transfection control,Renilla luciferase. pGL-3 (Promega) was used as the empty vector. Allthe experiments were performed four to eight times in triplicate(n=12-24).

FIG. 3D. The effects of miR-155 transfection in MEG-01 cells onexpression levels of uc.160 and uc.346A. Effects were measured byqRT-PCR at 0, 24 and 48 hours post-transfection.

FIG. 3E. Two scatter plots between expression values of mir-24-1 anduc.160 and of miR-155 and uc.346A are presented. The regression lineshows the negative correlation between these two genes. The name of thecorresponding array probes are presented on the Y and X axes. Bothprobes recognize the mature form of the miRNA gene.

FIGS. 4A-4D. T-UCR 73A(P) is acting as an oncogene in colon cancercells:

FIG. 4A. The expression inhibition by various siRNAs in COLO-320 cells.As reference value we used a siRNA control from Dharmacon. The mosteffective two siRNAs and a pool of four different siRNAs, includingthese two, were used.

FIG. 2B. The antiproliferative effects of reduction in uc.73A(P) geneexpression using siRNA-uc73A in COLO-320 colorectal cancer cells. Allthe results represent the median of three independent triplicateexperiments. The levels of uc. 73A (P) expression were measured byRT-PCR. Two asterisks indicate a statistically significant effect atP<0.01, while one at P<0.05.

FIG. 4C. Reduced levels of uc.73A(P) (using various siRNAs) results inenhanced apoptosis as shown by the Annexin-V staining assay in COLO-329cells. As reference value we used a siRNA control from Dharmacon.

FIG. 4D. Inhibition of uc.73A(P) by various siRNAs did not influenceSW620 colon cancer cell survival. All the results represent the medianof three independent triplicate experiments.

FIG. 5. UCR expression in human normal and malignant tissues by Northernblot. The expression for uc.192(N) and uc.246(E) in normal mononuclearcells (MNC) and CLL samples is presented. Normalization was done with U6probe. The arrows on the left side show the identified transcripts.Under the gel image there are the averages of UCR normalized expressionvalues in CLL and MNC samples from microarray experiments; p-values werefrom ANOVA statistic.

FIG. 6. UCR expression in human normal and malignant tissues by qRT-PCR.Relative gene expression by qRT-PCR in CD5+/CD19+ positive lymphocytesand human chronic lymphocytic leukemia (CLL) samples. UCR microarrayvalues of CLL and CD5+ samples are indicated under the graph; p-valueswere from ANOVA statistic.

FIG. 7. T-UCR expression profile of 22 normal human tissues. Clusteringof tissues and UCRs revealed a distinct pattern of UCRs expression innormal human tissues. Samples are shown in columns, T-UCRs in rows. Agreen colored gene is down-regulated compared to its median expressionin all samples, red is up-regulated and yellow means no variation.

FIG. 8. T-UCR expression profile of 173 cancers and corresponding normaltissues. Samples from leukemia and normal blood cells are separated fromcancer and tissue of epithelial origin. Samples are shown in columns,T-UCRs in rows. A green colored gene is down-regulated compared to itsmedian expression in all samples, red is up-regulated and yellow meansno variation.

FIG. 9. The expression of uc.73A(P) gene in various colon cancer celllines by quantitative RT-PCR. The expression in normal colon representsthe median value of 4 different samples. For normalization we usedbeta-actin.

FIG. 10. The uc.73A(P) inhibition by siRNA1 in COLO-320 and SW-620 cellsat 48 hrs. Comparable levels of inhibition in respect with a siRNA ofcontrol (Dharmacom) were achieved in both types of cells. In spite ofthis, the biological effects were seen only in COLO-320 cells, where theT-UCR is overexpressed about 2.5 times when compared with expression innormal colon.

FIG. 11. Downregulation by small interfering of uc.73A(P) inducesapoptosis in COLO-320 cells but not in SW-620 cells. Data obtained withcaspase-3 assay in COLO-320 (upper panel), where a significant increasein apoptotic cells is found, and in the control cells SW620 (lowerpanel), where no difference could be found. COLO-320 express high levelsof uc.73A(P), while in SW620 the expression is comparable with thenormal colon levels.

FIG. 12—Table 1. Most significant differentially expressed UCRs inleukemias and carcinomas.

FIG. 13—Table 2. Mixed effect Poisson regression results as associationof UCRs with regions of interest.

FIG. 14—Table 3. T-UCRs whose expression inversely correlates withcomplementary miRNA differentially expressed in CLL patients.

FIG. 15—Table 4. T-UCRs expression in 22 human normal tissues (3 induplicate, from 2 different individuals).

FIG. 16—Table 5. T-UCRs differentially expressed in CLL, CRC and HCCidentified by ANOVA analysis at P<0.005 (GeneSpring GX software).

FIG. 17—Table 6. Genomic location of UCRs is correlated with CAGR.(databases as in (Bejerano et al., 2004); (Cahn et al., 2004)).

FIG. 18—Table 7. Negative correlations between the expression of miRNAsand T-UCRs in CLL patients. All validated negative correlation by FDRmethod at 0.01 threshold, or 1% of false positive results, and with an Rcorrelation lower as 0.40 were considered.

DESCRIPTION OF EMBODIMENTS

As used herein, a “CAGR” includes any region of the genomic DNA thatcomprises a genetic or epigenetic change (or the potential for a geneticor epigenetic change) that differs from normal DNA, and which iscorrelated with a cancer. Exemplary genetic changes include single- anddouble-stranded breaks (including common breakpoint regions in or nearpossible oncogenes or tumor-suppressor genes); chromosomaltranslocations; mutations, deletions, insertions (including viral,plasmid or transposon integrations) and amplifications (including geneduplications) in the DNA; minimal regions of loss-of-heterozygosity(LOH) suggestive of the presence of tumor-suppressor genes; and minimalregions of amplification suggestive of the presence of oncogenes.Exemplary epigenetic changes include any changes in DNA methylationpatterns (e.g., DNA hyper- or hypo-methylation, especially in promoterregions).

Many of the known miR genes in the human genome are in or near CAGRs,including 80 miR genes that are located exactly in minimal regions ofLOH or minimal regions of amplification correlated to a variety ofcancers. Other miR genes are located in or near breakpoint regions,deleted areas, or regions of amplification.

For example, cancers associated with CAGRs include leukemia (e.g., AML,CLL, pro-lymphocytic leukemia), lung cancer (e.g., small cell andnon-small cell lung carcinoma), esophageal cancer, gastric cancer,colorectal cancer, brain cancer (e.g., astrocytoma, glioma,glioblastoma, medulloblastoma, meningioma, neuroblastoma), bladdercancer, breast cancer, cervical cancer, epithelial cancer,nasopharyngeal cancer (e.g., oral or laryngeal squamous cell carcinoma),lymphoma (e.g., follicular lymphoma), uterine cancer (e.g., malignantfibrous histiocytoma), hepatic cancer (e.g., hepatocellular carcinoma),head-and-neck cancer (e.g., head-and-neck squamous cell carcinoma),renal cancer, male germ cell tumors, malignant mesothelioma,myelodysplastic syndrome, ovarian cancer, pancreatic or biliary cancer,prostate cancer, thyroid cancer (e.g., sporadic follicular thyroidtumors), and urothelial cancer.

As used herein, a “FRA” includes any rare or common fragile site in achromosome; e.g., one that can be induced by subjecting a cell to stressduring DNA replication. For example, a rare FRA can be induced bysubjecting the cell to folic acid deficiency during DNA replication. Acommon FRA can be induced by treating the cell with aphidocolin or5-azacytidine during DNA replication. The identification or induction ofchromosomal fragile sites is within the skill in the art; see, e.g.,Arlt et al. (2003), Cytogenet. Genome Res. 100:92-100 and Arlt et al.(2002), Genes, Chromosomes and Cancer 33:82-92, the entire disclosuresof which are herein incorporated by reference.

Approximately 20% of the known human miR genes are located in (13 miRs)or within 3 Mb (22 miRs) of cloned FRAs. Indeed, the relative incidenceof miR genes inside fragile sites occurs at a rate 9.12 times higherthan in non-fragile sites. Moreover, after studying 113 fragile sites ina human karyotype, it was found that 61 miR genes are located in thesame chromosomal band as a FRA.

For example, cancers associated with FRAs include bladder cancer,esophageal cancer, lung cancer, stomach cancer, kidney cancer, cervicalcancer, ovarian cancer, breast cancer, lymphoma, Ewing sarcoma,hematopoietic tumors, solid tumors and leukemia.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Genome-wide profiling reveals extensive transcription of ultraconservedregions (UCRs) in normal human tissues.

To investigate the involvement of UCRs in human cancers, we analyzed 481genomic regions longer than 200 bp (Bejerano et al., 2004b) by Northernblot, quantitative PCR (qRT-PCR) and microarray.

Both exonic (E) and non-exonic (N) UCR probes detected transcripts (insense or antisense—A, orientation) over a large range of lengths fromvarious normal tissues (FIG. 1A and FIG. 5).

The length of two of the transcripts was confirmed by cloning the cDNAby 5′- and 3′-RACE for the exonic uc.246(E) from normal human colon andthe non-exonic uc.269A(N) from normal human bone marrow. Neither ofthese cDNAs contained open reading frames (ORFs) of significant length,confirming their likely non-protein coding nature. These non-splicedfull-length cDNAs, that we named non-coding ultraconserved genes,nc-UCGs, are of variable length (about 0.8 kb for the ultraconservedgene UCG.246 and about 1.8 kb and 2.8 kb for the ultraconserved geneUCG.269A).

Transcription of these nc-UCGs may be initiated from poly-adenine richgenomic regions, as was recently proposed for several long ncRNAs frommouse (Furuno et al., 2006).

We compared the transcription levels of several UCRs from normal anddisease tissue using microarray analysis followed by qRT-PCR andNorthern blot confirmation. The expression of uc.291(P) and uc. 73A(P)was significantly higher in normal CD5+/CD 19+ lymphocytes than in CLLcells (P<0.05) (FIG. 1B). The data obtained with this microarrayplatform has been confirmed in various studies (Cahn et al., 2005a;Yanaihara et al., 2006; Volinia et al., 2006).

The strength of our data is reinforced by the fact that two independentsets of normal CD5 cells were included in microarray and quantitativeRT-PCR experiments. When both uc.291(P) and uc.73A(P) were investigatedby qRT-PCR and microarray in two different sets of CD5/CD19 positive Bcells and malignant B cells, the differential expression wasstatistically significant by both assays (FIG. 1B).

Furthermore, qRT-PCR and Northern blotting for eleven and six UCRs,respectively, gave results that were concordant with microarray results(FIG. 5 and FIG. 6).

Using microarray analysis, we found that the majority of transcribedUCRs (that we named here T-UCRs) were expressed in normal human tissuesboth ubiquitously and in a tissue-specific manner (FIG. 1C).

About 34% of putative T-UCRs (325/962) had hybridization signals with anintensity over background (calculated as average signal of blank spots+2SD) in all 19 tissue samples. The highest number of T-UCRs was found inB cells, while the lowest was in ovary. About 93% of the UCRs (890 of962) were expressed over background in at least one sample, andtherefore we considered these as T-UCRs. The three different types ofUCRs were transcribed with similar frequencies: 41% of exonic UCRs, 33%of possibly exonic UCRs and 30% of non-exonic UCRs.

The microarray platform contains putative T-UCRs in both sense andanti-sense orientation. Eighty-four of the 962 UCRs (9%) werebidirectionally transcribed, while 241 were transcribed only from onestrand, in all the normal tissues analyzed (FIG. 1D, FIG. 1E and Table4).

Since identification of bidirectional transcription by microarrayanalysis may be hindered by trace contamination with genomic DNA, weperformed a comparison of microarray results with strand-specificqRT-PCR for uc.2 69(N), uc.233(E) and uc. 73(P). In all three instancesthe data were concordant, showing predominant transcription from onestrand (FIG. 1E).

Of note, out of the 156 non-exonic T-UCRs expressed in all 19 tissues,92 (−60%) are intergenic, while 64 are intronic. Of the latter, 37 arein the antisense orientation compared with the host gene, suggestingthat about 83% (129/156) of the non-exonic T-UCRs did not representintronic transcription of long precursor transcripts of known hostgenes, but bona fide independent noncoding transcripts.

As with miRNAs (Liu et al., 2004), we performed a hierarchicalclustering of T-UCR expression in hematopoietic tissues (represented byB lymphocytes, T lymphocytes and mononuclear cells, each collected fromtwo healthy individuals) and non-hematopoietic tissues. The same typesof tissue from different individuals were clustered as closest neighbors(FIG. 2A and FIG. 7).

These findings demonstrate that UCRs represent, in a significantproportion of cases, non-coding transcripts in normal human tissues andthat the expression of these T-UCRs is tissue-specific.

Distinct UCR Signatures in Human Leukemias and Carcinomas

Since extensive gene expression alterations in cancer cells have beenwidely described for both protein coding genes and miRNAs(Esquela-Kerscher and Slack, 2006; Calin and Croce, 2006a; Calin andCroce, 2006b; Lu et al., 2005), we investigated the expression of UCRsin a panel of 173 samples, including 133 human cancers and 40corresponding normal tissues.

Hierarchical clustering of the samples showed that various types ofcancers clustered differently according to their developmental origins:the leukemias (CLL) and normal hematopoietic tissues were branchedseparately from the colorectal (CRC) and hepatocellular carcinomas (HCC)with their normal counterparts (FIG. 8); moreover, specific groups ofUCRs seemed to be differentially expressed in tumor types (FIG. 2B).

Since different tissues have specific UCR signatures, this clusteringpattern could be the consequence of different tissue-specific origin ofthe tumors. Thus, we compared the expression of UCRs between the normaland tumor cells of the same origin. Out of 962 possible T-UCRs, 88(9.1%) were differentially expressed at a highly statisticallysignificant level (P<0.005) in at least one type of cancer (Table 1 andTable 5).

We found both down-regulated and up-regulated T-UCRs in cancers comparedto the expression in corresponding normal tissues. By comparing eachcancer type with the corresponding normal tissues, we found that the CLLsignature was composed of 19 UCRs (8 up- and 11 down-regulated), the CRCsignature of 61 UCRs (59 up- and 2 down-regulated), and the HCCsignature of 8 UCRs (3 up- and 5 down-regulated) (Table 5).

Eighteen transcripts of the signatures were exonic UCRs (20%), 28 werepossibly exonic UCRs (32%) and 42 were non-exonic UCRs (48%). Of the 18exonic T-UCRs, 9 represented the anti-sense direction of the known hostprotein-coding gene transcripts. We therefore demonstrated that theT-UCR expression profiles can be used to differentiate human cancers.

UCRs are Frequently Located at Fragile Sites and Genomic RegionsInvolved in Cancers

We compared the genomic location of UCRs with that of previouslyreported non-random genetic alterations identified in human tumors andcloned fragile sites (FRA) as described (Calin et al., 2004b). We usedthe set of 186 miRNAs previously reported (Cahn et al., 2004b) and a setof 297 zinc finger protein-coding genes (ZNF) (genome.ucsc.edu), a wellknown family of transcription factors shown to be associated with cancer(Huntley et al., 2006).

We previously reported that miRNA genes are frequently located at FRAsites, HOX genes clusters and genomic regions involved in cancer, suchas minimal regions of loss of heterozygosity (LOH), and minimal regionsof amplification, globally named cancer associated genomic regions(CAGR) (Calin et al., 2004b).

A recent study, using high-resolution array comparative genomichybridization (aCGH), confirmed that miRNA loci exhibit genomicalterations at high frequency in human cancers (Zhang and al, 2006).Furthermore, by analyzing the miRNA expression in NCl-60 cell lines,another group found that the candidate tumor-suppressor and oncogenicmiRNAs are located in CAGRs (Gaur et al, 2007).

Here, we show that the association between the genomic location of UCRsand the analyzed cancer-related genomic elements is highly statisticallysignificant and comparable to that reported for miRNAs. The ZNFtranscription factors did not show any significant association with anyof the analyzed regions of interest (Table 2 and Table 6).

There was a similar lack of association for the smaller family ofprotein-coding genes involved in RNA splicing (80 genes, data notshown). For example, the probability for the association of UCRs ormiRNAs with minimal LOH regions versus non-deleted genomic regions wasless than 0.001 in both instances (IRR of 2.02 and 4.08, respectively).As an internal control, we used the human papilloma virus 16 (HPV 16)integration sites, which frequently occur in FRA sites. If UCRs aresignificantly associated with FRA, then we expected to fin anassociation with the HPV 16 integration site. This is exactly what weobserved for both UCRs and miRNAs, but not for ZNF protein-coding genes(Table 2) or for the protein-coding genes involved in RNA splicing (datanot shown).

Additional data illustrate the importance of the genomic location ofUCRs. First, we found that the ubiquitously expressed T-UCRs (expressedin 18 or 19 normal tissues in FIG. 1C) are significantly more frequentlylocated in CAGRs (P<0.005, Fisher exact test) when compared with allother UCRs (97 out of 189 vs. 71 out of 292). Second, T-UCRsdifferentially expressed in human cancers are located in CAGRsspecifically associated with that type of cancer. For example, thechromosomal region 13g21.33-g22.2 has been linked to susceptibility tofamilial CLL (Ng et al, 2007). No mutations were found in any of the 13protein-coding genes screened within this interval.

We identified a cluster of seven UCRs (uc.347 to uc.353) located withinthis CAGR. Two of them, uc.349A(P) and uc.352(N) are among the T-UCRsthat are differentially expressed between normal and malignant B-CLL CD5positive cells.

This suggests, at least in this case, that it is not the protein-codinggenes but the UCRs that represent the “unknown” culprits located in theCAGR. Together these data provide evidence that the UCRs are located ingenomic regions altered during the malignant process, and suggest thatT-UCRs could be candidate genes for cancer susceptibility.

Negative Regulation of T-UCRs by Direct Interaction with MicroRNAs

In order to begin to functionally characterize some UCRs involved inhuman cancers, we performed a genome-wide expression study in the sameset of CLL samples investigated above. We found that a signature of fiveUCRs, uc.269A(N), uc.160(N), uc.215(N), uc.346A(P) and uc.348(N), wasable to differentiate between two main CLL prognosis groups previouslydifferentiated by the expression of 70-kDa zeta-associated protein(ZAP-70).

These five T-UCRs displayed variations in their expression level thatwas negatively correlated with the miRNA expression signature reportedin CLL (Cahn et al., 2005a) (Table 3).

While not wishing to be bound by theory, the inventor herein nowbelieves that this finding raises the possibility of complex regulatorymechanisms between miRNAs and T-UCRs. We identified, by sequencealignments, that three out of the 5 UCRs have significant antisensecomplementarity with 5 out of the 13 miRNAs from the signature, givingrise to six possible interacting pairs: uc.160:: miR-24, uc.160::miR-155, uc.160:: miR-223, uc.160::miR-146a, uc.346A::miR-155, anduc.-348::miR-29b (FIG. 3A).

In this analyzed set of miRNA::UCR pairs, the 5′-end “6 base seed”complementarity rule described for miRNA::mRNA interaction was valid;furthermore, the levels of 3′-end complementarity could be variable:more than 60% complementarity for miR-24:: uc160 or miR-155::uc.346Apairs to less than 25% for the miR-155:: uc.160 pair. As a control, whenrandomly generated combinations of five UCRs and 13 miRNAs werecompared, the sense and antisense complementarity was not significant.

Negative correlations between the microa7ay expression values ofspecific T-UCRs and predicted interactor miRNAs was confirmed by qRT-PCRfor selected T-UCRs and miRNAs from lymphocytes of an independent set ofCLL patients and normal controls (FIG. 3B).

We performed in vitro assays of miRNA::UCR interaction involving miR-155which is overexpressed in the aggressive form of CLL (Calin et al.,2005) some lymphomas and carcinomas (E is et al., 2005; Kluiver et al.,2005; Volinia et al., 2006), and miR-24-1 and miR29-b which carrymutations in primary transcripts from CLL patients (Calin et al.,2005a).

We cloned the UCRs uc.160(N), uc.346A(P) and uc.348(N) in luciferasereporter vectors to assess the possible direct interaction with miR-155,miR-24-1 or miR-29-b. We observed consistent and reproducible reductionin luciferase expression with four miR::T-UCR pairings consistent withinteractions taking place in vitro (FIG. 3C).

By contrast, uc.348(N) did not interact with miR155 as indicated by theluciferase assay, a result that is in concordance with the positiveexpression correlation of these two genes in CLL patients and the lowsequence complementarity (FIG. 3A).

Interactions In Vivo

In order to determine if these interactions occur in vivo, wetransfected miR-155 into MEG01 leukemia cells and assessed the levels ofuc.346A and uc.160 (both well expressed in this cell line). At 24 hoursafter transfection, miR-155 significantly reduced the expression levelof both T-UCRs; after 48 hours, the reduction of exogenous miR-155levels was paralleled by an increase in T-UCR expression (FIG. 3D).

This reversible effect supports a regulation of T-UCR by specificmiRNAs. As this interaction was proven for the genes of the “ZAP-70signature”, we investigated the correlations between the expression ofall miRNAs and T-UCRs at the genome-wide level in all 50 CLL patients.Interestingly, we found a significant negative correlation (at a falsedetection rate (FDR) of less than 0.01) between 87 miRNAs (out of 235spotted on the chip, 37%) and T-UCRs expression levels (Table 7).

Furthermore, among the correlated genes we identified the miR-24-1::uc.160 and the miR155:: uc346A(P) pairs, experimentally proven tointeract (FIG. 3E).

Moreover, miR-155 and uc.348, that did not interact experimentally, werenot members of this list. Other pairs of possible interactors for whichwe identified positive luciferase assays were miR-15-a:: uc.78 andmiR16:: uc. 78 (data not shown). Therefore, non-coding T-UCRs representpossible targets of miRNAs, and these interactions may have biologicaland prognostic significance for cancer patients.

T-UCRs May Act as Oncogenes

To expand the functional characterization of T-UCR, we examined thebiological effects of uc. 73A (P) in a cancer model. Since this is oneof the most statistically significant up-regulated T-UCRs in coloncancers (P<0.001), we decided to investigate the effects of itsdownregulation in COLO-320 colorectal cancer cells that expressed highlevels of uc.73A(P). As a control we used the SW620 colon cancer cellsin which the expression of this gene does not differ from normal coloniccells (FIG. 9).

Two small interfering RNAs (siRNA1 and siRNA3), as well as a pool offour siRNAs (siRNApool), were designed to target uc. 73A(P) andtransfected into COLO-320 and SW620 cells. There was significantly lessexpression of uc.73A(P) after 48 (FIG. 4A and FIG. 10), 72 and 144 hours(data not shown) in the COLO-320 cells treated with siRNAs 1, 3 andpool. The same was found also for SW-620 cells (FIG. 6).

The growth of COLO-320 cells was significantly reduced after 144 hoursof treatment with specific siRNA compared to both untreated (null) orsiRNA-treated control cells (P<0.05 at 96 hrs and P<0.005 at 144 hrs)(FIG. 4B).

In comparison, proliferation of the SW620 control cells was notsignificantly changed (P=0.83 and P=0.23 at 96 and 144 hrs,respectively) (data not shown). Cell cycle studies revealed an increasein the sub-G1 fraction of cells (suggesting the presence of apoptoticcells, data not shown) in COLO-320 cells, but not in SW620 cells, afinding confirmed by the apoptosis-specific AnnexinV assay (FIG. 4C andFIG. 4D) and by caspase-3 assay (FIG. 11).

Furthermore, the intensity of effects on cell proliferation and survivalwere proportional with the degree of inhibition by siRNAs (FIG. 4).

These data suggest that in colorectal cancers, uc. 73A(P) behaves likean oncogene by increasing the number of malignant cells as a consequenceof reduced apoptosis.

DISCUSSION

According to the dogma of molecular oncology, cancer is a geneticdisease involving tumor-suppressor and oncogenic proteins (Bishop, 1991;Hunter, 1991; Weinberg, 1991). Recent findings strongly support theinvolvement of microRNAs in the pathogenesis of a majority of analyzedcancers, and add a new layer of complexity to the molecular architectureof human cancers (Calin et al., 2002; Esquela-Kerscher and Slack, 2006;Calin and Croce, 2006a). MiRNAs represent, however, just a particulargroup of ncRNAs involved in human cancers. It has been shown thatantisense intronic ncRNA levels correlate with the degree of tumordifferentiation in prostate cancer (Reis et al., 2005) and that MALAT-1ncRNA expression predicts metastasis and survival in early stagenon-small cell lung cancer (Ji et al., 2003), suggesting a deeper linkbetween ncRNAs and tumor biology.

To clearly address this question, we investigated at the genomic level afull new class of ncRNAs, namely the transcribed non-codingultraconserved regions (T-UCRs). We used bioinformatics tools todemonstrate that the UCRs are located in genomic regions targeted duringthe malignant process indicative of a putative involvement in humantumorigenesis.

As now shown herein, we were able to clone by RACE amplification cDNAscorresponding to uc.246(E) and uc.269A(N), proving that the UCRs arebona fide genes (named herein as nc-UCGs) that are expressed and can becloned by standard methods.

Various expression techniques including Northern blot, qRT-PCR andgenome-wide microarray profiling, proved that UCRs are frequentlytranscribed and that there are distinct signatures in human leukemiasand carcinomas. We focused on chronic lymphocytic leukemia, the mostfrequent adult leukemia in the Western world (Chiorazzi et al., 2005),on colorectal carcinoma, one of the most common cancers inindustrialized countries (de la Chapelle, 2004) and on hepatocellularcarcinoma, the most rapidly increasing type of cancer in America(Wilson, 2005).

We found that for all the tumor types examined, the malignant cells havea unique spectrum of expressed UCRs when compared with the correspondingnormal cells, suggesting that significant variations in T-UCR expressionare involved in the malignant process.

Characterizing the functional significance of T-UCR alterations in humancancers is not a trivial task. A myriad of putative functions of T-UCRscan be hypothesized, including an antisense inhibitory role for proteincoding genes or other non-codingRNAs, or a role as “aspecific” miRNAs,meaning miRNAs with peculiarities such as very long precursors (e.g.uc.339(P) which has a precursor length that is double the usual miRNA).This puzzle becomes more complicated by the fact that several UCRs donot act like genes and were found to have regulatory functions asenhancers (Nobrega et al., 2003; Pennacchio et al., 2006), while othersrepresent exons of protein coding genes with known/unknown cancerconnections. A particularly interesting region is the DA CHI locus thatcontains 7 UCRs in about 700 kb (Bejerano et al., 2004b). Three of theUCRs from this region are differentially expressed in analyzed cancers,two of which are members of the CLL signature. The majority of scannedconserved regions from this locus in a mouse model are enhancers,including the uc.351(N) that was not expressed in any of the analyzedtissues in our study.

Interestingly, the only two regions that failed to have enhancerfunction are uc.348(N) and uc.352(N), both classified as non-coding andboth differentially expressed in human cancers. Further increasing theinterest in these specific T-UCRs, is the finding that this genomicregion has been linked to susceptibility to familial CLL and that noneof the known protein-coding genes were mutated (Ng et al, 2007).

Recently, it was found that short blocks of several tens of by from thenoncoding parts of the human genome (named pyknons) occur within nearlyall known protein coding genes (Rigoutsos et al., 2006). While thepyknons are distinct from the UCRs, the ultraconserved elementcontaining the highest number of pyknons (four) was uc. 73(P), which wefound to be one of the most differentially expressed T-UCRs in both CLLand CRC. These intriguing observations suggest a possible regulatoryrole for uc. 73(P) on the coding genes with complementary sequences.

Further expanding the involvement of this T-UCR in human cancers, wewere able to prove an oncogenic function for uc.73(P) in colon cancer,as diminution of its over-expression induced apoptosis and hadantiproliferative effects specifically in colon cancer cells abnormallyexpressing this T-UCR.

Our findings that another class of ncRNAs, the T-UCRs, is consistentlyaltered at the genomic level in a high percentage of analyzed leukemiasand carcinomas, support a model in which both coding and non-codinggenes are involved and cooperate in human tumorigenesis (Calin andCroce, 2006b).

Furthermore, correlations between the expression of UCRs and miRNAs inCLL patients raise the intriguing possibility of complex functionalregulatory pathways in which two or more types of ncRNAs interact andinfluence the phenotype.

We also demonstrated the existence of the miRNA::T-UCR interaction inwhich two different types of ncRNAs are interacting.

We found that nc-UCGs are consistently altered at the genomic level in ahigh percentage of leukemias and carcinomas, and may interact withmiRNAs in leukemias. The findings provide support for a model in whichboth coding and non-coding genes are involved in and cooperate in humantumorigenesis.

Example I Experimental Procedures

A) RACE Cloning and Expression Analysis by Microarray, qRT-PCR andNorthern Blot

1) RACE Cloning

The expression of six UCRs (uc.47(N), uc.110(N), uc.192(N), uc.246(E),uc.269A(N) and uc.352(N)) was analyzed in brain, testis, bone marrow,small intestine, colon and liver tissue using various combinations ofPCR primers designed to amplify short products. These products included40-mers used for probes in microarray analysis and the complete >200 bpUCR sequence. Two of the UCR products, one exonic, uc.246(E) and onenon-exonic, uc.269A(N), were cloned by Rapid Amplification of cDNA Ends(RACE) in both 5′ and 3′ directions. The sources of tissue from whichsequences were cloned were bone marrow, leukocytes, fetal brain andcolon according to the manufacturer protocol (Marathon-ready cDNAs,Clontech, Palo Alto, Calif.).

2) UCR Expression Study by Microarray.

Total RNA was extracted with Trizol (Invitrogen, Carlsbad, Calif.) from19 normal human tissues (Liu et al., 2004) and from 50 CLL samples frompatients diagnosed with CLL. Informed consent was obtained from allpatients at the CLL Research Consortium institutions in the US. Ascontrols, CDS+ B cells from 6 healthy individuals (four distinctsamples, two being pools from two different healthy individuals) andmononuclear cells (MNC) from 3 individuals were used as reported in(Calin et al., 2005a). RNA was also extracted from 78 primary colorectalcarcinomas, 21 normal colonic mucosas, 9 primary hepatocellularcarcinomas and 4 normal livers, collected at the University of Ferrara,University of Bologna and University Tor Vergata, Rome (Italy). Allsamples were obtained with written informed consent according toinstitutional guidelines for the protection of human subjects.

Microarray chips were developed with a total of 481 human UCR sequencesas in (soe.ucsc.edu/-jill/ultra). For each UCR two 40-mer probes weredesigned, one corresponding to the sense genomic sequence (named “+”)and the other to the complementary sequence (named “A+”). The designcriteria were as described (Liu et al., 2004). Each oligo was printed induplicate in two different slide locations, and therefore quadruplicatenumerical values were available for analysis. Several thousand (3484)blank spots were used for background subtraction. RNA extraction andmicroarray experiments, consisting of the UCR microarray assembly,target preparation and array hybridization, were performed as describedin detail elsewhere (Liu et al., 2004; Calin et al., 2004a).

Briefly, 5 μg of RNA from each tissue sample was labeled with biotin byreverse transcription using random hexamers. Hybridization was carriedout on the second version of our miRNA-chip (ArrayExpress accessionnumber: A-MEXP-258) which contained the 962 UCR probes, 238 probes formature miRNA and 143 probes for precursor miRNAs. Each oligo was printedin duplicate in two different slide locations. Hybridization signalswere detected by biotin binding of a Streptavidin-Alexa647 conjugate(one-color signal) using a GenePix 4000B scanner (Axon Instruments).Images were quantified using the GenePix Pro 6.0 (Axon Instruments).

Raw data were normalized and analyzed in GeneSpring GX 7.3 (AgilentTechnologies, Santa Clara, Calif.). Expression data of the 22 tissuesamples were normalized with Lowess function in Bioconductor (Limmapackage) and then were median centered using GeneSpring normalization;the threshold used to determine the level of UCR expression wascalculated as the average of blank spots+2 SD (standard deviation).Tumors were normalized using the on-chip and on-gene mediannormalization of the GeneSpring software. Hierarchical cluster analysiswas done using average linkage and Pearson correlation as measures ofsimilarity. Statistical comparisons of tumors and normal tissues wereperformed by filtering on fold change and then using the ANOVA (Analysisof Variance) statistic of the GeneSpring software and the Benjamin andHochberg correction for reduction of false positives. The filter onfold-change was set on 1.2 because this threshold, already used formicroRNAs analyzed with the same chip [see for examples (Cahn et al.,2005a; Cimmino et al., 2005; Iorio et al., 2005)], was demonstrated toreflect a real biological difference. The T-UCRs differentiallyexpressed among CLL patients, grouped in accordance to 70-kDazeta-associated protein (ZAP-70) expression, were identified bycombining the ANOVA results with the SAM (Significance Analysis ofMicroarray) and PAM (Prediction Analysis of Microarrays) analysis. Theirexpression was compared to that of microRNAs (Calin et al., 2005a). Alldata were submitted using MIAMExpress to the ArrayExpress database andcould be retrieve using the accession number E-TABM-184.

3) Quantitative RT-PCR for UCRs.

Quantitative RT-PCR was the first method we used to confirm themicroarray results. We validated the microarray data for eleven UCRs,including uc. 73 (P)/73A (P), uc.135(E), uc.160(N), uc.233(E)/233A(E),uc.269(N)/269A(N), uc.289(N), uc.291(P), and uc.346A(P) in variouscombinations of samples, including 15 to 17 randomly selected CLLsamples from the array set of 50, and various normal CD 19+/CD5+ B cellsand B and T lymphocyte controls by qRT-PCR. An additional set of 3normal CD19+/CD5+ positive B cells, not used for microarray studies, waspurchased from AllCells (Berkeley, Calif.), and used as an independentconfirmation set. In all instances the qRT-PCR data confirmed themicroarray data. RNA was treated with RNase-free DNase I and reversetranscribed to cDNA using random primers and SuperScript II reversetranscriptase. To determine if the sense or antisense UCR transcript wasexpressed, total RNA was reverse transcribed using Thermoscript RT and agene specific (i.e. sense or antisense) primer. RT conditions were asdescribed (Schmittgen et al., 2004). cDNA was amplified using real-timePCR and SYBR green detection using PCR primers designed to amplify thesame 40 by regions as the oligo probe on the microarray. The relativeamount of each UCR to 18S rRNA was determined using the equation 2″dcT,where dCT=(CTUCR−CT18s rRNA). Relative gene expression data weremultiplied by 106 to simplify the presentation.

4) Northern Blot Analysis of T-UCRs.

We analyzed five UCRs, uc.110(N), uc.192(N), uc.246(E), uc.269A(N) anduc.352(N), by Northern blot, two of which were then cloned by RACEexperiments. For a sixth one, the uc. 47(N), the data are not shown.Total RNA was electrophoresed on 15% PAA-urea gels (Calin et al., 2002).RNA sources included 11 normal tissues (breast, liver, lung, kidney andpancreas) in duplicate or triplicate (purchased from Ambion andClontech) and 4 normal MNC samples and 16 CLL samples prepared in thelaboratory. As this represents the investigation by Northern blot of UCRexpression, we used multiple samples from the same tissues to confirmdata reproducibility. The probes were designed to be identical with theoligonucleotides on the chip in order to detect the same transcripts asthe microarray, and the hybridization was done as described (Cahn etal., 2002).

B) Databases and Statistical Analyses.

1) Databases for Genomic Locations.

The UCR databases used for all the studies reported here are aspublished (Bejerano et al., 2004b). We restricted our analyses to 481segments longer than 200 base pairs (bp). The Fragile site (FRA)database and the cancer associated genomic regions (CAGR) databases areas previously published (Cain et al., 2004b).

2) Statistical Analyses for Genomic Locations.

To test hypotheses associating the incidence of ultra-conserved regions(UCRs) with fragile sites, amplified regions in cancer, and deletionregions in cancer, we utilized random effect Poisson and negativebinomial regression models. Under these models, “events” were defined asthe number of UCRs, and exposure “time” (i.e. fragile site versusnon-fragile site) was defined as non-overlapping lengths of the regionof interest. The “length” of a region was exact, if known, or estimatedas 1 Mb if unknown. For example, for each chromosome the total length ofall non-overlapping fragile sites was computed and was used as theexposure time for fragile sites. We then counted the number of UCRsoccurring within fragile sites for each chromosome. The remaining lengthof each chromosome (total Mb−fragile sites Mb) was assumed to benon-fragile, and the remaining UCRs in each chromosome were assumed tooccur in the non-fragile region. Then for each region, alternativerandom effects models, the zero-inflated Poisson and the zero-inflatednegative binomial models were fitted, and, of the three, the best modelwas selected using the Akaike's Information Criteria (based on the loglikelihood and number of parameters). This same approach was used foranalysis of the data from expression of zinc finger proteins. The bestfitting model for fragile sites with UCRs and LOH with zinc fingerproteins was the zero-inflated negative binomial. For all other cases,the Poisson model is reported. When the number of categories with zeroevents was more than expected for a Poisson distribution, thezero-inflated negative binomial model was preferred. When the totalnumber of events was too small for a region, the model likelihoods wereunable to converge, and results are not reported. The random effect inthe Poisson, zero-inflated Poisson and zero-inflated negative binomialregression models, was the individual chromosome, in that data within achromosome was assumed to be correlated. The fixed effect in each modelconsisted of an indicator variable(s) for the type of region beingcompared. We report the incidence rate ratio (IRR), 2-sided 95%confidence interval of the incidence rate ratio, and 2-sided p-valuesfor testing the hypothesis that the incident rate ratio is 1.0. An IRRsignificantly>1 indicates an increase in the number of UCRs within aregion over that expected by chance.

The proportions of clustering of miRNAs and zinc finger proteins werecompared using an asymptotic test of the difference in two independentproportions, where we report the difference, 95% confidence interval ofthe difference, and p-value. Of note, the ZNF transcription factor classof genes showed a significantly lower clustering (a cluster defined asthe location of at least two genes from the same class at less than 50kb genomic distance) when compared with the microRNAs (32%, 95/297clustered ZNF genes versus 48%, 90/186 clustered miRNAs,difference=16.4%, 95% CI=(7.5%, 25.2%), P<0.001). All computations werecompleted using STATA v7.0 and StatXact v7.0.

Statistical Analyses for Negative Correlations Between MicroarrayExpression of UCRs and miRNAs.

A detailed description is provided in the Example II and data therein.Briefly, the input data was constituted by a list of T-UCRs and by alist of miRNAs (the “seeds”) and the corresponding matrix of expressionvalues. We calculated r, the Spearman rank coefficient of correlationfor each pair of (miR, UC) genes; namely, we evaluate the P-values ofthe correlation tests and select the genes whose correlation value issignificant at a given value of rejection. Given the high number ofcorrelation tests performed, P-values were corrected for multipletesting by using the false detection rate (FDR), as in (Benjamini andHochberg, 1995). In this way, P-values control the number of falsepositive over the number of truly null tests, while FDR controls thenumber of false positive over the number of significant tests.

C) Functional Studies

1) UCRs Down-Regulation by Direct Interaction with MicroRNAs.

The genomic sequences of uc.160, uc.346A and uc.348 were cloned intopGL3-control vector (Clontech) using the XbaI site immediatelydownstream from the stop codon of luciferase. Human megakaryocyticMEG-O1 and the cervical carcinoma HeLa cell lines were grown asrecommended by the ATCC. Cells were co-transfected in 12-well platesusing siPORT neoFX (Ambion) according to the manufacturer's protocolusing 0.4 μg of the firefly luciferase reporter vector and 0.08 μg ofthe control vector containing Renilla luciferase, pRL-TK (Promega). Foreach well 10 nM of miRNA-sense precursor and scrambled oligonucleotides(Ambion) were used. Firefly and Renilla luciferase activities weremeasured consecutively using the Dual-luciferase assays (Promega) 24 hrafter transfection. All experiments were performed in triplicate on fourto six different days (n=12 to 18).

Expression of both the ultraconserved RNA and the mature miRNA wasanalyzed using real-time PCR. Expression of the UCR RNA was determinedby real-time PCR as described above. Expression of the mature miRNA wasperformed using TaqMan looped primer assays to miR-155 (AppliedBiosystems) as described (Chen et al., 2005). Mature miRNA expressionwas presented as 2-dCT where dCT=CTmiRNA−CT1 ss rRNA); data wasmultiplied by 106 to simplify presentation.

For the patient correlation a set of 13 samples was used (including 9CLL patients and 4 normal lymphocyte samples) and miR-155, uc.346A anduc.160 levels were analyzed as described herein. For the identificationof the “in vivo” effects in MEGO1 of miR-155 transfection, the levels ofuc.346A and uc.160 were measured by qRT-PCR as described at 0, 24 and 48hrs post-transfection with the pre-miRNA 155 (Ambion) usingLipofectamine reagent.

2) Effects on Cancer Cell Proliferation by uc. 73A(P) Inhibition.

The siRNA against the uc. 73A(P) were designed using the Dharmaconalgorithm (Dharmacon siDESIGN (harmacon.com/sidesign)) entering thecomplete sequence of the UCR. The eight highest rank target sequenceswere tested. The performance was assessed after 48, 72 and 144 hourpost-transfection by semi-quantitative RT-PCR. The most effective twosiRNAs and a pool of four different siRNAs, including these two, wereused. We named these as siRNA1, siRNA3 and siRNApool. For the cellgrowth assay, the human colon cancer cell lines COLO-320 and SW620 weregrown in RPMI1640 medium supplemented with 10% FBS and 1×10⁴ cells wereplated in 96-well plate a day before transfection. The cells weretransfected with siRNA-uc.73A(P) at a final concentration of 200 nM byusing Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA) accordingto the manufacturer's protocol. The siCONTROL Non-Targeting siRNA Pool(Dharmacon Research, LaFayette, Colo., USA) was used as negativecontrol. The transfection was repeated under the same conditions everytwo days, at 48 and 96 hour. To evaluate the cell number the CellTiter96 Aqueous One Solution Cell Proliferation Assay (Promega U.S., Madison,Wis., USA) was used. The readings were performed at 0, 48, 96, and 144hours, respectively measuring the absorbance at 490 nm using an ELISAplate reader (Spectra MAX, Molecular Devices, Sunnyvale, Calif., USA).The cell growth assay was performed three times in triplicate for eachtreatment. The statistical differences between the number of cells atvarious time points with respect to time 0, was calculated using the ttest.

For both cell cycle and apoptosis assays, cells were plated in 6 wellplates at 6×105 cells per well. The day after and then every 48 hrs, thecells were transfected with 200 nM siRNA. The cells were collected andfixed in cold 70% ethanol for at least 30 minutes. The Propidium Iodide(PI) staining was performed at 48, 96, and 144 hours in a 50 μg/mL PI(Sigma Aldrich, St. Louis, Mo.) and 5.1 g/mL RNAse DNAse free (RocheDiagnostics, Indianapolis, Ind., USA) PBS Solution. The apoptosisstaining was performed with the Annexin V-FITC Apoptosis Detection Kit(BD Pharmingen, San Jose, Calif., USA) and with the PE-conjugatedmonoclonal active Caspase-3 antibody apoptosis kit (BD Biosciences) at 0and 144 hours according to the manufacturer's procedure using an FACSCalibur (BD Biosciences, San Jose, Calif., USA) to acquire the data.Each experiment was performed three times.

The GeneBank accession numbers for the cloned T-UCRs described in thisstudy are as followings: DQ644536 (UCG.246), DQ644537 (UCG.269A, shortform), and DQ644538 (UCG.269A, long form).

Example II Experimental Procedures

Statistical Analyses for Negative Correlations Between MicroarrayExpression of UCRs and miRNAs.

The input data were constituted by a list of UCGs and by a list ofmiRNAs (the “seeds”) and the corresponding matrix of expression values.We calculated r, the Spearman rank coefficient of correlation, anon-parametric measure of data trend correlation based on rankings, foreach pair of (miR, UCR) genes; namely, we evaluate the P-values of thecorrelation tests and selected the genes whose correlation value issignificant at a given value of rejection. Evaluation of P-values wasperformed assuming that the correlation values are distributed usingStudent's t cumulative distribution, with a number of degrees of freedomcorresponding to the number of samples in the microarray experiment. TheP-values measure the ‘goodness’ of the single correlations (amongcouples of genes), therefore, to understand if the real correlationderives by chance or represents a biologically important information, wechoose the method of permutations, changing the order of the samples foreach row (miR or UCR) and calculating the correlations between pair ofgenes (miR, UCR) with different changed samples orders. We repeated thesamples permutation and computed correlations 100 times, in this way,every real correlation has 100 random correlations to compare with.Using all (100*n° MIR*n° UC) random correlations and real correlations,we recalculated P-values based on random correlations ranking andposition of the real correlations.

Given the high number of correlation tests performed, P-values werecorrected for multiple testing by using the false detection rate (FDR),as defined by (Benjamini and Hochberg, 1995). In this way, P-valuescontrol the number of false positive over the number of truly nulltests, while FDR controls the number of false positive over the numberof significant tests. Several ways of estimating this number have beenproposed, and we adopted the solution devised by Tom Nichols (seefroi.sourceforge.net/documents/technical/matlab/FDR), that rescales theP-value obtained on a single test multiplying it by a combination ofindexes related to the total number of tests performed. Correction wasperformed on a seed by seed basis, meaning that the genes in the seedslist were considered independent tests. This statistically validatedtripe filtering allows the targeted extraction of a shortlist ofcandidate genes, thus saving resources for the following costly andtime-consuming genetic analysis.

To build a scatter plot between miR-24-1 and uc.160 expression values,we plotted a regression line by using MatLab function ROBUSTFIT toexplain hypotheses of negative correlation between these two genes.Notice that only 11.67% (7/60) of points (pairs of expression values)are outlier.

Example III Additional Examples and Information

As used herein miR and UCRs are used interchangeably; including in anon-limiting manner: a “miR gene product,” “microRNA,” “miR,” or “miRNA”refers to the unprocessed (e.g., precursor) or processed (e.g., mature)RNA transcript from a miR gene.

Diagnosis Using UCRs (miRNAs)

In one aspect, there is provided herein methods of diagnosing whether asubject has, or is at risk for developing, a cancer, comprisingmeasuring the level of at least one UCR in a test sample from thesubject and comparing the level of the miR gene product in the testsample to the level of a corresponding miR gene product in a controlsample. As used herein, a “subject” can be any mammal that has, or issuspected of having, a cancer. In a preferred embodiment, the subject isa human who has, or is suspected of having, a cancer.

The level of at least one miR gene product can be measured in abiological sample (e.g., cells, tissues) obtained from the subject. Forexample, a tissue sample (e.g., from a tumor) can be removed from asubject suspected of having a cancer-related disease by conventionalbiopsy techniques. In another embodiment, a blood sample can be removedfrom the subject, and blood cells (e.g., white blood cells) can beisolated for DNA extraction by standard techniques. The blood or tissuesample is preferably obtained from the subject prior to initiation ofradiotherapy, chemotherapy or other therapeutic treatment. Acorresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR gene product produced from a given miR gene incells from the subject's sample can be compared to the corresponding miRgene product levels from cells of the control sample. A reference miRexpression standard for the biological sample can also be used as acontrol.

An alteration (e.g., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of a cancer-related disease in the subject.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “up-regulated”). As used herein, expression of a miR gene product is“up-regulated” when the amount of miR gene product in a cell or tissuesample from a subject is greater than the amount of the same geneproduct in a control cell or tissue sample.

In another embodiment, the level of the at least one miR gene product inthe test sample is less than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “down-regulated”). As used herein, expression of a miR gene is“down-regulated” when the amount of miR gene product produced from thatgene in a cell or tissue sample from a subject is less than the amountproduced from the same gene in a control cell or tissue sample.

The relative miR gene expression in the control and normal samples canbe determined with respect to one or more RNA expression standards. Thestandards can comprise, for example, a zero miR gene expression level,the miR gene expression level in a standard cell line, the miR geneexpression level in unaffected tissues of the subject, or the averagelevel of miR gene expression previously obtained for a population ofnormal human controls.

The level of a miR gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques (e.g., Northern blot analysis,RT-PCR, in situ hybridization) for determining RNA expression levels ina biological sample (e.g., cells, tissues) are well known to those ofskill in the art. In a particular embodiment, the level of at least onemiR gene product is detected using Northern blot analysis. For example,total cellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7, the entire disclosure of whichis incorporated by reference.

Suitable probes for Northern blot hybridization of a given miR geneproduct can be produced from the nucleic acid sequences and include, butare not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or complete complementarity to a miR gene product ofinterest. Methods for preparation of labeled DNA and RNA probes, and theconditions for hybridization thereof to target nucleotide sequences, aredescribed in Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters10 and 11, the disclosures of which are incorporated herein byreference.

In one non-limiting example, the nucleic acid probe can be labeled with,e.g., a radionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal;a ligand capable of functioning as a specific binding pair member for alabeled ligand (e.g., biotin, avidin or an antibody); a fluorescentmolecule; a chemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl) deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference.

In one non-limiting example, suitable probes for in situ hybridizationof a given miR gene product can be produced from the nucleic acidsequences, and include, but are not limited to, probes having at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarityto a miR gene product of interest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods forperforming quantitative and semi-quantitative RT-PCR, and variationsthereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer. Assessing cancer-specific expression levels for hundredsof miR genes or gene products is time consuming and requires a largeamount of total RNA (e.g., at least 20 μg for each Northern blot) andautoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotides) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level incancer cells.

As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from cancerous (e.g., tumor)tissue, and within cancerous tissue, different prognosis states (forexample, good or poor long term survival prospects) may be determined.By comparing expression profiles of the cancer tissue in differentstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.The identification of sequences that are differentially expressed incancer tissue, as well as differential expression resulting in differentprognostic outcomes, allows the use of this information in a number ofways.

In one non-limiting example, a particular treatment regime may beevaluated (e.g., to determine whether a chemotherapeutic drug acts toimprove the long-term prognosis in a particular patient). Similarly,diagnosis may be done or confirmed by comparing patient samples withknown expression profiles. Furthermore, these gene expression profiles(or individual genes) allow screening of drug candidates that suppressthe cancer expression profile or convert a poor prognosis profile to abetter prognosis profile.

Accordingly, there is also provided herein methods of diagnosing whethera subject has, or is at risk for developing, a cancer, comprisingreverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample,and comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample or reference standard, whereinan alteration in the signal of at least one miRNA is indicative of thesubject either having, or being at risk for developing, cancer.

In one embodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina particular embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR29a, miR-29b, miR-29c and combinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT (TrisHCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the arraywhere the immobilized probe DNA recognizes a complementary target cDNAin the sample, hybridization occurs. The labeled target cDNA marks theexact position on the array where binding occurs, allowing automaticdetection and quantification. The output consists of a list ofhybridization events, indicating the relative abundance of specific cDNAsequences, and therefore the relative abundance of the correspondingcomplementary miRs, in the patient sample.

According to one embodiment, the labeled cDNA oligomer is abiotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool allows for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a cancer-relateddisease quantitatively reverse transcribed to provide a set of labeledtarget oligodeoxynucleotides complementary to the RNA in the sample. Thetarget oligodeoxynucleotides are then hybridized to a microarraycomprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal).

More preferably, the profile recorded includes the intensity of thesignal from each hybridization. The profile is compared to thehybridization profile generated from a normal, i.e., noncancerous,control sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

There is also provided herein methods of determining the prognosis of asubject with a cancer, comprising measuring the level of at least onemiR gene product, which is associated with a particular prognosis in acancer-related disease (e.g., a good or positive prognosis, a poor oradverse prognosis), in a test sample from the subject.

According to these methods, an alteration in the level of a miR geneproduct that is associated with a particular prognosis in the testsample, as compared to the level of a corresponding miR gene product ina control sample, is indicative of the subject having a cancer with aparticular prognosis. In one embodiment, the miR gene product isassociated with an adverse (i.e., poor) prognosis. Examples of anadverse prognosis include, but are not limited to, low survival rate andrapid disease progression. In certain embodiments, the level of the atleast one miR gene product is measured by reverse transcribing RNA froma test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to amicroarray that comprises miRNA-specific probe oligonucleotides toprovide a hybridization profile for the test sample, and comparing thetest sample hybridization profile to a hybridization profile generatedfrom a control sample.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of cancers. Therefore, alteringthe level of the miR gene product (e.g., by decreasing the level of amiR gene product that is up-regulated in cancer cells, by increasing thelevel of a miR gene product that is down-regulated in cancer cells) maysuccessfully treat the cancer.

Accordingly, there is further provided herein methods of inhibitingtumorigenesis in a subject who has, or is suspected of having, a cancerwherein at least one miR gene product is deregulated (e.g.,down-regulated, up-regulated) in the cancer cells of the subject. Whenthe at least one isolated miR gene product is down-regulated in thecancer cells (e.g., miR-29 family), the method comprises administeringan effective amount of the at least one isolated miR gene product, or anisolated variant or biologically-active fragment thereof, such thatproliferation of cancer cells in the subject is inhibited.

For example, when a miR gene product is down-regulated in a cancer cellin a subject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to the endogenous wild-type miR gene product (e.g., a miR geneproduct) that is down-regulated in the cancer cell or it can be avariant or biologically-active fragment thereof.

As defined herein, a “variant” of a miR gene product refers to a miRNAthat has less than 100% identity to a corresponding wild-type miR geneproduct and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e.g., inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with cancer (e.g., cell differentiation, cell growth,cell death). These variants include species variants and variants thatare the consequence of one or more mutations (e.g., a substitution, adeletion, an insertion) in a miR gene. In certain embodiments, thevariant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to a corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with acancer. In certain embodiments, the biologically-active fragment is atleast about 5, 7, 10, 12, 15, or 17 nucleotides in length.

In a particular embodiment, an isolated miR gene product can beadministered to a subject in combination with one or more additionalanti-cancer treatments. Suitable anti-cancer treatments include, but arenot limited to, chemotherapy, radiation therapy and combinations thereof(e.g., chemoradiation).

When the at least one isolated miR gene product is up-regulated in thecancer cells, the method comprises administering to the subject aneffective amount of at least one compound for inhibiting expression ofthe at least one miR gene product, referred to herein as miR geneexpression-inhibition compounds, such that proliferation of the cancercells is inhibited. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene productselected from the group consisting miR29 family, including miR-29a,miR-29b, miR-29c, and combinations thereof.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, a cancer, including preventing or delaying the onset of thedisease symptoms, and/or lessening the severity or frequency of symptomsof the disease or condition. The terms “subject”, “patient” and“individual” are defined herein to include animals, such as mammals,including, but not limited to, primates, cows, sheep, goats, horses,dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine,equine, canine, feline, rodent, or murine species. In a preferredembodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from a cancer. One skilled in the art can readilydetermine an effective amount of a miR gene product to be administeredto a given subject, by taking into account factors, such as the size andweight of the subject; the extent of disease penetration; the age,health and sex of the subject; the route of administration; and whetherthe administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product is administered to a subject can range fromabout 5 to about 3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in substantially-purified form, or can exist in acell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to one particular embodiment,the isolated miR gene products described herein can be used for themanufacture of a medicament for treating a cancer in a subject (e.g., ahuman).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in a cancer cell,using, for example, the techniques for determining miR transcript leveldiscussed above for the diagnostic method. Inhibition can occur at thelevel of gene expression (i.e., by inhibiting transcription of a miRgene encoding the miR gene product) or at the level of processing (e.g.,by inhibiting processing of a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer (e.g., a cancer). One skilledin the art can readily determine an effective amount of a miRexpression-inhibition compound to be administered to a given subject, bytaking into account factors, such as the size and weight of the subject;the extent of disease penetration; the age, health and sex of thesubject; the route of administration; and whether the administration isregional or systemic.

For example, an effective amount of the expression-inhibition compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and interfere with the expression of (e.g., inhibit translationof, induce cleavage or destruction of) the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product.

Without wishing to be bound by any theory, it is believed that theantisense nucleic acids activate RNase H or another cellular nucleasethat digests the miR gene product/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups.

Exemplary enzymatic nucleic acids for use in the present methods includede novo methyltransferases, including DNMT3A and DNMT3B, as described inthe Examples herein.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al.(1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No.4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a cancer.

As used herein, to “inhibit the proliferation of a cancer cell” means tokill the cell, or permanently or temporarily arrest or slow the growthof the cell. Inhibition of cancer cell proliferation can be inferred ifthe number of such cells in the subject remains constant or decreasesafter administration of the miR gene products or miR geneexpression-inhibition compounds. An inhibition of cancer cellproliferation can also be inferred if the absolute number of such cellsincreases, but the rate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibition compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression-inhibition compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds.

In one embodiment, the cells are transfected with a plasmid or viralvector comprising sequences encoding at least one miR gene product ormiR gene expression-inhibition compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-E1-[2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

A miR gene product or miR gene expression-inhibition compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibition compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene productexpression-inhibition compound. Suitable delivery reagents include,e.g., the Mirus Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine), andliposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibition compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibition compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization-inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating a cancer.

In one embodiment, the pharmaceutical composition comprises at least oneisolated miR gene product, or an isolated variant or biologically-activefragment thereof, and a pharmaceutically-acceptable carrier. In aparticular embodiment, the at least one miR gene product corresponds toa miR gene product that has a decreased level of expression in cancercells relative to suitable control cells.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound. In aparticular embodiment, the at least one miR gene expression-inhibitioncompound is specific for a miR gene whose expression is greater incancer cells than control cells.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% byweight), or a physiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical compositions of the invention additionally comprise oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising sequences encoding them), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is one or more of miR29a, miR-29b and miR-29c.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) that is resistant to degradation by nucleases.

One skilled in the art can readily synthesize nucleic acids that arenuclease resistant, for example, by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

Pharmaceutical Compositions

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising sequences encoding them) encapsulated in a liposome asdescribed above, and a propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel, FOLFOX4.

Inhibitors of Tumorigenesis

There is also provided herein methods of identifying an inhibitor oftumorigenesis, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in cancer cells. An increase in the level ofthe miR gene product in the cell after the agent is provided, relativeto a suitable control cell (e.g., agent is not provided), is indicativeof the test agent being an inhibitor of tumorigenesis.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in cancer cells. A decrease in thelevel of the miR gene product in the cell after the agent is provided,relative to a suitable control cell (e.g., agent is not provided), isindicative of the test agent being an inhibitor of tumorigenesis. In aparticular embodiment, at least one miR gene product associated withincreased expression levels in cancer cells is selected from the groupconsisting of miR29a, miR-29b, miR-29c, and combinations thereof.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described hereinabove.

Example IV Methods, Reagents and Kits for Diagnosing, Staging,Prognosing, Monitoring and Treating Cancer-Related Diseases

It is to be understood that all examples herein are to be considerednon-limiting in their scope. Various aspects are described in furtherdetail in the following subsections.

Diagnostic Methods

In one embodiment, there is provided a diagnostic method of assessingwhether a patient has a cancer-related disease or has higher than normalrisk for developing a cancer-related disease, comprising the steps ofcomparing the level of expression of a marker in a patient sample andthe normal level of expression of the marker in a control, e.g., asample from a patient without a cancer-related disease.

A significantly higher level of expression of the marker in the patientsample as compared to the normal level is an indication that the patientis afflicted with a cancer-related disease or has higher than normalrisk for developing a cancer-related disease.

The markers are selected such that the positive predictive value of themethods is at least about 10%, and in certain non-limiting embodiments,about 25%, about 50% or about 90%. Also preferred for use in the methodsare markers that are differentially expressed, as compared to normalcells, by at least two-fold in at least about 20%, and in certainnon-limiting embodiments, about 50% or about 75%.

In one diagnostic method of assessing whether a patient is afflictedwith a cancer-related disease (e.g., new detection (“screening”),detection of recurrence, reflex testing), the method comprisescomparing: a) the level of expression of a marker in a patient sample,and b) the normal level of expression of the marker in a controlnon-cancer-related disease sample. A significantly higher level ofexpression of the marker in the patient sample as compared to the normallevel is an indication that the patient is afflicted with acancer-related disease.

There is also provided diagnostic methods for assessing the efficacy ofa therapy for inhibiting a cancer-related disease in a patient. Suchmethods comprise comparing: a) expression of a marker in a first sampleobtained from the patient prior to providing at least a portion of thetherapy to the patient, and b) expression of the marker in a secondsample obtained from the patient following provision of the portion ofthe therapy. A significantly lower level of expression of the marker inthe second sample relative to that in the first sample is an indicationthat the therapy is efficacious for inhibiting a cancer-related diseasein the patient.

It will be appreciated that in these methods the “therapy” may be anytherapy for treating a cancer-related disease including, but not limitedto, pharmaceutical compositions, gene therapy and biologic therapy suchas the administering of antibodies and chemokines. Thus, the methodsdescribed herein may be used to evaluate a patient before, during andafter therapy, for example, to evaluate the reduction in disease state.

In certain aspects, the diagnostic methods are directed to therapy usinga chemical or biologic agent. These methods comprise comparing: a)expression of a marker in a first sample obtained from the patient andmaintained in the presence of the chemical or biologic agent, and b)expression of the marker in a second sample obtained from the patientand maintained in the absence of the agent. A significantly lower levelof expression of the marker in the second sample relative to that in thefirst sample is an indication that the agent is efficacious forinhibiting a cancer-related disease in the patient. In one embodiment,the first and second samples can be portions of a single sample obtainedfrom the patient or portions of pooled samples obtained from thepatient.

Methods for Assessing Prognosis

There is also provided a monitoring method for assessing the progressionof a cancer-related disease in a patient, the method comprising: a)detecting in a patient sample at a first time point, the expression of amarker; b) repeating step a) at a subsequent time point in time; and c)comparing the level of expression detected in steps a) and b), andtherefrom monitoring the progression of a cancer-related disease in thepatient. A significantly higher level of expression of the marker in thesample at the subsequent time point from that of the sample at the firsttime point is an indication that the cancer-related disease hasprogressed, whereas a significantly lower level of expression is anindication that the cancer-related disease has regressed.

There is further provided a diagnostic method for determining whether acancer-related disease has worsened or is likely to worsen in thefuture, the method comprising comparing: a) the level of expression of amarker in a patient sample, and b) the normal level of expression of themarker in a control sample. A significantly higher level of expressionin the patient sample as compared to the normal level is an indicationthat the cancer-related disease has worsened or is likely to worsen inthe future.

Methods for Assessing Inhibitory, Therapeutic and/or HarmfulCompositions

There is also provided a test method for selecting a composition forinhibiting a cancer-related disease in a patient. This method comprisesthe steps of: a) obtaining a sample comprising cells from the patient;b) separately maintaining aliquots of the sample in the presence of aplurality of test compositions; c) comparing expression of a marker ineach of the aliquots; and d) selecting one of the test compositionswhich significantly reduces the level of expression of the marker in thealiquot containing that test composition, relative to the levels ofexpression of the marker in the presence of the other test compositions.

There is additionally provided a test method of assessing the harmfulpotential of a compound in causing a cancer-related disease. This methodcomprises the steps of: a) maintaining separate aliquots of cells in thepresence and absence of the compound; and b) comparing expression of amarker in each of the aliquots. A significantly higher level ofexpression of the marker in the aliquot maintained in the presence ofthe compound, relative to that of the aliquot maintained in the absenceof the compound, is an indication that the compound possesses suchharmful potential.

In addition, there is further provided a method of inhibiting acancer-related disease in a patient. This method comprises the steps of:a) obtaining a sample comprising cells from the patient; b) separatelymaintaining aliquots of the sample in the presence of a plurality ofcompositions; c) comparing expression of a marker in each of thealiquots; and d) administering to the patient at least one of thecompositions which significantly lowers the level of expression of themarker in the aliquot containing that composition, relative to thelevels of expression of the marker in the presence of the othercompositions.

The level of expression of a marker in a sample can be assessed, forexample, by detecting the presence in the sample of: the correspondingmarker protein or a fragment of the protein (e.g. by using a reagent,such as an antibody, an antibody derivative, an antibody fragment orsingle-chain antibody, which binds specifically with the protein orprotein fragment) the corresponding marker nucleic acid (e.g. anucleotide transcript, or a complement thereof), or a fragment of thenucleic acid (e.g. by contacting transcribed polynucleotides obtainedfrom the sample with a substrate having affixed thereto one or morenucleic acids having the entire or a segment of the nucleic acidsequence or a complement thereof) a metabolite which is produceddirectly (i.e., catalyzed) or indirectly by the corresponding markerprotein.

Any of the aforementioned methods may be performed using at least one ora plurality (e.g., 2, 3, 5, or 10 or more) of cancer-related diseasemarkers. In such methods, the level of expression in the sample of eachof a plurality of markers, at least one of which is a marker, iscompared with the normal level of expression of each of the plurality ofmarkers in samples of the same type obtained from control humans notafflicted with a cancer-related disease. A significantly altered (i.e.,increased or decreased as specified in the above-described methods usinga single marker) level of expression in the sample of one or moremarkers, or some combination thereof, relative to that marker'scorresponding normal or control level, is an indication that the patientis afflicted with a cancer-related disease. For all of theaforementioned methods, the marker(s) are selected such that thepositive predictive value of the method is at least about 10%.

Examples of Candidate Agents

The candidate agents may be pharmacologic agents already known in theart or may be agents previously unknown to have any pharmacologicalactivity. The agents may be naturally arising or designed in thelaboratory. They may be isolated from microorganisms, animals or plants,or may be produced recombinantly, or synthesized by any suitablechemical method. They may be small molecules, nucleic acids, proteins,peptides or peptidomimetics. In certain embodiments, candidate agentsare small organic compounds having a molecular weight of more than 50and less than about 2,500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins. Candidateagents are also found among biomolecules including, but not limited to:peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. There are, for example,numerous means available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. In certain embodiments, thecandidate agents can be obtained using any of the numerous approaches incombinatorial library methods art, including, by non-limiting example:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection.

In certain further embodiments, certain pharmacological agents may besubjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

The same methods for identifying therapeutic agents for treating acancer-related disease can also be used to validate leadcompounds/agents generated from in vitro studies.

The candidate agent may be an agent that up- or down-regulates one ormore cancer-related disease response pathways. In certain embodiments,the candidate agent may be an antagonist that affects such pathway.

Methods for Treating a Cancer-Related Disease

There is provided herein methods for treating, inhibiting, relieving orreversing a cancer-related disease response. In the methods describedherein, an agent that interferes with a signaling cascade isadministered to an individual in need thereof, such as, but not limitedto, cancer-related disease patients in whom such complications are notyet evident and those who already have at least one cancer-relateddisease response.

In the former instance, such treatment is useful to prevent theoccurrence of such cancer-related disease response and/or reduce theextent to which they occur. In the latter instance, such treatment isuseful to reduce the extent to which such cancer-related diseaseresponse occurs, prevent their further development or reverse thecancer-related disease response.

In certain embodiments, the agent that interferes with thecancer-related disease response cascade may be an antibody specific forsuch response.

Expression of Markers

Expression of a marker can be inhibited in a number of ways, including,by way of a non-limiting example, an antisense oligonucleotide can beprovided to the cancer-related disease cells in order to inhibittranscription, translation, or both, of the marker(s). Alternately, apolynucleotide encoding an antibody, an antibody derivative, or anantibody fragment which specifically binds a marker protein, andoperably linked with an appropriate promoter/regulator region, can beprovided to the cell in order to generate intracellular antibodies whichwill inhibit the function or activity of the protein. The expressionand/or function of a marker may also be inhibited by treating thecancer-related disease cell with an antibody, antibody derivative orantibody fragment that specifically binds a marker protein. Using themethods described herein, a variety of molecules, particularly includingmolecules sufficiently small that they are able to cross the cellmembrane, can be screened in order to identify molecules which inhibitexpression of a marker or inhibit the function of a marker protein. Thecompound so identified can be provided to the patient in order toinhibit cancer-related disease cells of the patient.

Any marker or combination of markers, as well as any certain markers incombination with the markers, may be used in the compositions, kits andmethods described herein. In general, it is desirable to use markers forwhich the difference between the level of expression of the marker incancer-related disease cells and the level of expression of the samemarker in normal cells is as great as possible. Although this differencecan be as small as the limit of detection of the method for assessingexpression of the marker, it is desirable that the difference be atleast greater than the standard error of the assessment method, and, incertain embodiments, a difference of at least 2-, 3-, 4-, 5-, 6-, 7-,8-, 9-, 10-, 15-, 20-, 100-, 500-, 1000-fold or greater than the levelof expression of the same marker in normal tissue.

It is recognized that certain marker proteins are secreted to theextracellular space surrounding the cells. These markers are used incertain embodiments of the compositions, kits and methods, owing to thefact that such marker proteins can be detected in a cancer-associatedbody fluid sample, which may be more easily collected from a humanpatient than a tissue biopsy sample. In addition, in vivo techniques fordetection of a marker protein include introducing into a subject alabeled antibody directed against the protein. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques.

In order to determine whether any particular marker protein is asecreted protein, the marker protein is expressed in, for example, amammalian cell, such as a human cell line, extracellular fluid iscollected, and the presence or absence of the protein in theextracellular fluid is assessed (e.g. using a labeled antibody whichbinds specifically with the protein).

It will be appreciated that patient samples containing tissue and/orfluid cells may be used in the methods described herein. In theseembodiments, the level of expression of the marker can be assessed byassessing the amount (e.g., absolute amount or concentration) of themarker in a sample. The cell sample can, of course, be subjected to avariety of post-collection preparative and storage techniques (e.g.,nucleic acid and/or protein extraction, fixation, storage, freezing,ultrafiltration, concentration, evaporation, centrifugation, etc.) priorto assessing the amount of the marker in the sample.

It will also be appreciated that the markers may be shed from the cellsinto the digestive system, the blood stream and/or interstitial spaces.The shed markers can be tested, for example, by examining the serum orplasma.

The compositions, kits and methods can be used to detect expression ofmarker proteins having at least one portion which is displayed on thesurface of cells which express it. For example, immunological methodsmay be used to detect such proteins on whole cells, or computer-basedsequence analysis methods may be used to predict the presence of atleast one extracellular domain (i.e., including both secreted proteinsand proteins having at least one cell-surface domain). Expression of amarker protein having at least one portion which is displayed on thesurface of a cell which expresses it may be detected without necessarilylysing the cell (e.g., using a labeled antibody which binds specificallywith a cell-surface domain of the protein).

Expression of a marker may be assessed by any of a wide variety ofmethods for detecting expression of a transcribed nucleic acid orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of secreted, cell-surface, cytoplasmic or nuclearproteins, protein purification methods, protein function or activityassays, nucleic acid hybridization methods, nucleic acid reversetranscription methods and nucleic acid amplification methods.

In a particular embodiment, expression of a marker is assessed using anantibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a marker protein or fragment thereof,including a marker protein which has undergone all or a portion of itsnormal post-translational modification.

In another particular embodiment, expression of a marker is assessed bypreparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in apatient sample, and by hybridizing the mRNA/cDNA with a referencepolynucleotide which is a complement of a marker nucleic acid, or afragment thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide; preferably, it is not amplified.Expression of one or more markers can likewise be detected usingquantitative PCR to assess the level of expression of the marker(s).Alternatively, any of the many methods of detecting mutations orvariants (e.g., single nucleotide polymorphisms, deletions, etc.) of amarker may be used to detect occurrence of a marker in a patient.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker nucleic acid. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g., detectable using different chromophores orfluorophores, or fixed to different selected positions), then the levelsof expression of a plurality of markers can be assessed simultaneouslyusing a single substrate (e.g., a “gene chip” microarray ofpolynucleotides fixed at selected positions). When a method of assessingmarker expression is used which involves hybridization of one nucleicacid with another, it is desired that the hybridization be performedunder stringent hybridization conditions.

Biomarker Assays

In certain embodiments, the biomarker assays can be performed using massspectrometry or surface plasmon resonance. In various embodiment, themethod of identifying an agent active against al cancer-related diseasecan include a) providing a sample of cells containing one or moremarkers or derivative thereof; b) preparing an extract from said cells;c) mixing said extract with a labeled nucleic acid probe containing amarker binding site; and, d) determining the formation of a complexbetween the marker and the nucleic acid probe in the presence or absenceof the test agent. The determining step can include subjecting saidextract/nucleic acid probe mixture to an electrophoretic mobility shiftassay.

In certain embodiments, the determining step comprises an assay selectedfrom an enzyme linked immunoabsorption assay (ELISA), fluorescence basedassays and ultra high throughput assays, for example surface plasmonresonance (SPR) or fluorescence correlation spectroscopy (FCS) assays.In such embodiments, the SPR sensor is useful for direct real-timeobservation of biomolecular interactions since SPR is sensitive tominute refractive index changes at a metal-dielectric surface. SPR is asurface technique that is sensitive to changes of 10⁵ to 10⁻⁶ refractiveindex (RI) units within approximately 200 nm of the SPR sensor/sampleinterface. Thus, SPR spectroscopy is useful for monitoring the growth ofthin organic films deposited on the sensing layer.

Because the compositions, kits, and methods rely on detection of adifference in expression levels of one or more markers, it is desiredthat the level of expression of the marker is significantly greater thanthe minimum detection limit of the method used to assess expression inat least one of normal cells and cancer-affected cells.

It is understood that by routine screening of additional patient samplesusing one or more of the markers, it will be realized that certain ofthe markers are over-expressed in cells of various types, includingspecific cancer-related diseases.

In addition, as a greater number of patient samples are assessed forexpression of the markers and the outcomes of the individual patientsfrom whom the samples were obtained are correlated, it will also beconfirmed that altered expression of certain of the markers are stronglycorrelated with a cancer-related disease and that altered expression ofother markers are strongly correlated with other diseases. Thecompositions, kits, and methods are thus useful for characterizing oneor more of the stage, grade, histological type, and nature of acancer-related disease in patients.

When the compositions, kits, and methods are used for characterizing oneor more of the stage, grade, histological type, and nature of acancer-related disease in a patient, it is desired that the marker orpanel of markers is selected such that a positive result is obtained inat least about 20%, and in certain embodiments, at least about 40%, 60%,or 80%, and in substantially all patients afflicted with acancer-related disease of the corresponding stage, grade, histologicaltype, or nature. The marker or panel of markers invention can beselected such that a positive predictive value of greater than about 10%is obtained for the general population (in a non-limiting example,coupled with an assay specificity greater than 80%).

When a plurality of markers are used in the compositions, kits, andmethods, the level of expression of each marker in a patient sample canbe compared with the normal level of expression of each of the pluralityof markers in non-cancer samples of the same type, either in a singlereaction mixture (i.e. using reagents, such as different fluorescentprobes, for each marker) or in individual reaction mixturescorresponding to one or more of the markers. In one embodiment, asignificantly increased level of expression of more than one of theplurality of markers in the sample, relative to the corresponding normallevels, is an indication that the patient is afflicted with acancer-related disease. When a plurality of markers is used, 2, 3, 4, 5,8, 10, 12, 15, 20, 30, or 50 or more individual markers can be used; incertain embodiments, the use of fewer markers may be desired.

In order to maximize the sensitivity of the compositions, kits, andmethods (i.e. by interference attributable to cells of non-tissue and/orfluid origin in a patient sample), it is desirable that the marker usedtherein be a marker which has a restricted tissue distribution, e.g.,normally not expressed in a non-tissue cells.

It is recognized that the compositions, kits, and methods will be ofparticular utility to patients having an enhanced risk of developing acancer-related disease and their medical advisors. Patients recognizedas having an enhanced risk of developing a cancer-related diseaseinclude, for example, patients having a familial history of acancer-related disease.

The level of expression of a marker in normal human cells can beassessed in a variety of ways. In one embodiment, this normal level ofexpression is assessed by assessing the level of expression of themarker in a portion of cells which appear to be normal and by comparingthis normal level of expression with the level of expression in aportion of the cells which is suspected of being abnormal. Alternately,and particularly as further information becomes available as a result ofroutine performance of the methods described herein, population-averagevalues for normal expression of the markers may be used. In otherembodiments, the “normal” level of expression of a marker may bedetermined by assessing expression of the marker in a patient sampleobtained from a non-cancer-afflicted patient, from a patient sampleobtained from a patient before the suspected onset of a cancer-relateddisease in the patient, from archived patient samples, and the like.

There is also provided herein compositions, kits, and methods forassessing the presence of cancer-related disease cells in a sample (e.g.an archived tissue sample or a sample obtained from a patient). Thesecompositions, kits, and methods are substantially the same as thosedescribed above, except that, where necessary, the compositions, kits,and methods are adapted for use with samples other than patient samples.For example, when the sample to be used is a parafinized, archived humantissue sample, it can be necessary to adjust the ratio of compounds inthe compositions, in the kits, or the methods used to assess levels ofmarker expression in the sample.

Methods of Producing Antibodies

There is also provided herein a method of making an isolated hybridomawhich produces an antibody useful for assessing whether a patient isafflicted with a cancer-related disease. In this method, a protein orpeptide comprising the entirety or a segment of a marker protein issynthesized or isolated (e.g. by purification from a cell in which it isexpressed or by transcription and translation of a nucleic acid encodingthe protein or peptide in vivo or in vitro). A vertebrate, for example,a mammal such as a mouse, rat, rabbit, or sheep, is immunized using theprotein or peptide. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the protein or peptide, sothat the vertebrate exhibits a robust immune response to the protein orpeptide. Splenocytes are isolated from the immunized vertebrate andfused with an immortalized cell line to form hybridomas, using any of avariety of methods. Hybridomas formed in this manner are then screenedusing standard methods to identify one or more hybridomas which producean antibody which specifically binds with the marker protein or afragment thereof. There is also provided herein hybridomas made by thismethod and antibodies made using such hybridomas.

Methods of Assessing Efficacy

There is also provided herein a method of assessing the efficacy of atest compound for inhibiting cancer-related disease cells. As describedherein, differences in the level of expression of the markers correlatewith the abnormal state of the cells. Although it is recognized thatchanges in the levels of expression of certain of the markers likelyresult from the abnormal state of the cells, it is likewise recognizedthat changes in the levels of expression of other of the markers induce,maintain, and promote the abnormal state of those cells. Thus, compoundswhich inhibit a cancer-related disease in a patient will cause the levelof expression of one or more of the markers to change to a level nearerthe normal level of expression for that marker (i.e. the level ofexpression for the marker in normal cells).

This method thus comprises comparing expression of a marker in a firstcell sample and maintained in the presence of the test compound andexpression of the marker in a second cell sample and maintained in theabsence of the test compound. A significantly reduced expression of amarker in the presence of the test compound is an indication that thetest compound inhibits a cancer-related disease. The cell samples may,for example, be aliquots of a single sample of normal cells obtainedfrom a patient, pooled samples of normal cells obtained from a patient,cells of a normal cell line, aliquots of a single sample ofcancer-related disease cells obtained from a patient, pooled samples ofcancer-related disease cells obtained from a patient, cells of acancer-related disease cell line, or the like.

In one embodiment, the samples are cancer-related disease cells obtainedfrom a patient and a plurality of compounds believed to be effective forinhibiting various cancer-related diseases are tested in order toidentify the compound which is likely to best inhibit the cancer-relateddisease in the patient.

This method may likewise be used to assess the efficacy of a therapy forinhibiting a cancer-related disease in a patient. In this method, thelevel of expression of one or more markers in a pair of samples (onesubjected to the therapy, the other not subjected to the therapy) isassessed. As with the method of assessing the efficacy of testcompounds, if the therapy induces a significantly lower level ofexpression of a marker then the therapy is efficacious for inhibiting acancer-related disease. As above, if samples from a selected patient areused in this method, then alternative therapies can be assessed in vitroin order to select a therapy most likely to be efficacious forinhibiting a cancer-related disease in the patient.

Methods for Assessing Harmful Potentials

As described herein, the abnormal state of human cells is correlatedwith changes in the levels of expression of the markers. There is alsoprovided a method for assessing the harmful potential of a testcompound. This method comprises maintaining separate aliquots of humancells in the presence and absence of the test compound. Expression of amarker in each of the aliquots is compared. A significantly higher levelof expression of a marker in the aliquot maintained in the presence ofthe test compound (relative to the aliquot maintained in the absence ofthe test compound) is an indication that the test compound possesses aharmful potential. The relative harmful potential of various testcompounds can be assessed by comparing the degree of enhancement orinhibition of the level of expression of the relevant markers, bycomparing the number of markers for which the level of expression isenhanced or inhibited, or by comparing both.

Isolated Proteins and Antibodies

One aspect pertains to isolated marker proteins and biologically activeportions thereof, as well as polypeptide fragments suitable for use asimmunogens to raise antibodies directed against a marker protein or afragment thereof. In one embodiment, the native marker protein can beisolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, a protein or peptide comprising the whole or a segment ofthe marker protein is produced by recombinant DNA techniques.Alternative to recombinant expression, such protein or peptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”).

When the protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When the protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of the protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the polypeptide of interest.

Biologically active portions of a marker protein include polypeptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the marker protein, which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of a marker protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the markerprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the nativeform of the marker protein. In certain embodiments, useful proteins aresubstantially identical (e.g., at least about 40%, and in certainembodiments, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the correspondingnaturally-occurring marker protein yet differ in amino acid sequence dueto natural allelic variation or mutagenesis.

In addition, libraries of segments of a marker protein can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variant marker proteins or segments thereof.

Predictive Medicine

There is also provided herein uses of the animal models and markers inthe field of predictive medicine in which diagnostic assays, prognosticassays, pharmacogenomics, and monitoring clinical trials are used forprognostic (predictive) purposes to thereby treat an individualprophylactically. Accordingly, there is also provided herein diagnosticassays for determining the level of expression of one or more markerproteins or nucleic acids, in order to determine whether an individualis at risk of developing a cancer-related disease. Such assays can beused for prognostic or predictive purposes to thereby prophylacticallytreat an individual prior to the onset of the cancer-related disease.

In another aspect, the methods are useful for at least periodicscreening of the same individual to see if that individual has beenexposed to chemicals or toxins that change his/her expression patterns.

Yet another aspect pertains to monitoring the influence of agents (e.g.,drugs or other compounds administered either to inhibit a cancer-relateddisease or to treat or prevent any other disorder (e.g., in order tounderstand any system effects that such treatment may have) on theexpression or activity of a marker in clinical trials.

Pharmacogenomics

The markers are also useful as pharmacogenomic markers. As used herein,a “pharmacogenomic marker” is an objective biochemical marker whoseexpression level correlates with a specific clinical drug response orsusceptibility in a patient. The presence or quantity of thepharmacogenomic marker expression is related to the predicted responseof the patient and more particularly the patient's tumor to therapy witha specific drug or class of drugs. By assessing the presence or quantityof the expression of one or more pharmacogenomic markers in a patient, adrug therapy which is most appropriate for the patient, or which ispredicted to have a greater degree of success, may be selected.

Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof expression of a marker can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent to affect marker expression can be monitored in clinicaltrials of subjects receiving treatment for a cancer-related disease.

In one non-limiting embodiment, the present invention provides a methodfor monitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate) comprising the steps of(i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression ofone or more selected markers in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression of the marker(s) in thepost-administration samples; (v) comparing the level of expression ofthe marker(s) in the pre-administration sample with the level ofexpression of the marker(s) in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly.

For example, increased expression of the marker gene(s) during thecourse of treatment may indicate ineffective dosage and the desirabilityof increasing the dosage. Conversely, decreased expression of the markergene(s) may indicate efficacious treatment and no need to change dosage.

Electronic Apparatus Readable Media, Systems, Arrays and Methods ofUsing Same

As used herein, “electronic apparatus readable media” refers to anysuitable medium for storing, holding or containing data or informationthat can be read and accessed directly by an electronic apparatus. Suchmedia can include, but are not limited to: magnetic storage media, suchas floppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as compact disc; electronic storage media such asRAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybridsof these categories such as magnetic/optical storage media. The mediumis adapted or configured for having recorded thereon a marker asdescribed herein.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any method for recording information onmedia to generate materials comprising the markers described herein.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. Any number of data processor structuring formats (e.g.,text file or database) may be employed in order to obtain or create amedium having recorded thereon the markers. By providing the markers inreadable form, one can routinely access the marker sequence informationfor a variety of purposes. For example, one skilled in the art can usethe nucleotide or amino acid sequences in readable form to compare atarget sequence or target structural motif with the sequence informationstored within the data storage means. Search means are used to identifyfragments or regions of the sequences which match a particular targetsequence or target motif.

Thus, there is also provided herein a medium for holding instructionsfor performing a method for determining whether a subject has acancer-related disease or a pre-disposition to a cancer-related disease,wherein the method comprises the steps of determining the presence orabsence of a marker and based on the presence or absence of the marker,determining whether the subject has a cancer-related disease or apre-disposition to a cancer-related disease and/or recommending aparticular treatment for a cancer-related disease or pre-cancer-relateddisease condition.

There is also provided herein an electronic system and/or in a network,a method for determining whether a subject has a cancer-related diseaseor a pre-disposition to a cancer-related disease associated with amarker wherein the method comprises the steps of determining thepresence or absence of the marker, and based on the presence or absenceof the marker, determining whether the subject has a cancer-relateddisease or a pre-disposition to a cancer-related disease, and/orrecommending a particular treatment for the cancer-related disease orpre-cancer-related disease condition. The method may further comprisethe step of receiving phenotypic information associated with the subjectand/or acquiring from a network phenotypic information associated withthe subject.

Also provided herein is a network, a method for determining whether asubject has a cancer-related disease or a pre-disposition to acancer-related disease associated with a marker, the method comprisingthe steps of receiving information associated with the marker, receivingphenotypic information associated with the subject, acquiringinformation from the network corresponding to the marker and/or acancer-related disease, and based on one or more of the phenotypicinformation, the marker, and the acquired information, determiningwhether the subject has a cancer-related disease or a pre-disposition toa cancer-related disease. The method may further comprise the step ofrecommending a particular treatment for the cancer-related disease orpre-cancer-related disease condition.

There is also provided herein a business method for determining whethera subject has a cancer-related disease or a pre-disposition to acancer-related disease, the method comprising the steps of receivinginformation associated with the marker, receiving phenotypic informationassociated with the subject, acquiring information from the networkcorresponding to the marker and/or a cancer-related disease, and basedon one or more of the phenotypic information, the marker, and theacquired information, determining whether the subject has acancer-related disease or a pre-disposition to a cancer-related disease.The method may further comprise the step of recommending a particulartreatment for the cancer-related disease or pre-cancer-related diseasecondition.

Arrays

There is also provided herein an array that can be used to assayexpression of one or more genes in the array. In one embodiment, thearray can be used to assay gene expression in a tissue to ascertaintissue specificity of genes in the array. In this manner, up to about7000 or more genes can be simultaneously assayed for expression. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative determination, there is provided hereinthe quantitation of gene expression. Thus, not only tissue specificity,but also the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined.

Such a determination is useful, for example, to know the effect ofcell-cell interaction at the level of gene expression. If an agent isadministered therapeutically to treat one cell type but has anundesirable effect on another cell type, the method provides an assay todetermine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a cancer-related disease, progression of a cancer-relateddisease, and processes, such as cellular transformation associated witha cancer-related disease.

The array is also useful for ascertaining the effect of the expressionof a gene or the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

Surrogate Markers

The markers may serve as surrogate markers for one or more disorders ordisease states or for conditions leading up to a cancer-related diseasestate. As used herein, a “surrogate marker” is an objective biochemicalmarker which correlates with the absence or presence of a disease ordisorder, or with the progression of a disease or disorder. The presenceor quantity of such markers is independent of the disease. Therefore,these markers may serve to indicate whether a particular course oftreatment is effective in lessening a disease state or disorder.Surrogate markers are of particular use when the presence or extent of adisease state or disorder is difficult to assess through standardmethodologies, or when an assessment of disease progression is desiredbefore a potentially dangerous clinical endpoint is reached.

The markers are also useful as pharmacodynamic markers. As used herein,a “pharmacodynamic marker” is an objective biochemical marker whichcorrelates specifically with drug effects. The presence or quantity of apharmacodynamic marker is not related to the disease state or disorderfor which the drug is being administered; therefore, the presence orquantity of the marker is indicative of the presence or activity of thedrug in a subject. For example, a pharmacodynamic marker may beindicative of the concentration of the drug in a biological tissue, inthat the marker is either expressed or transcribed or not expressed ortranscribed in that tissue in relationship to the level of the drug. Inthis fashion, the distribution or uptake of the drug may be monitored bythe pharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.

Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker transcription orexpression, the amplified marker may be in a quantity which is morereadily detectable than the drug itself. Also, the marker may be moreeasily detected due to the nature of the marker itself; for example,using the methods described herein, antibodies may be employed in animmune-based detection system for a protein marker, or marker-specificradiolabeled probes may be used to detect a mRNA marker. Furthermore,the use of a pharmacodynamic marker may offer mechanism-based predictionof risk due to drug treatment beyond the range of possible directobservations.

Protocols for Testing

The method of testing for cancer-related diseases comprises, for examplemeasuring the expression level of each marker gene in a biologicalsample from a subject over time and comparing the level with that of themarker gene in a control biological sample.

When the marker gene is one of the genes described herein and theexpression level is differentially expressed (for examples, higher orlower than that in the control), the subject is judged to be affectedwith a cancer-related disease. When the expression level of the markergene falls within the permissible range, the subject is unlikely to beaffected with a cancer-related disease.

The standard value for the control may be pre-determined by measuringthe expression level of the marker gene in the control, in order tocompare the expression levels. For example, the standard value can bedetermined based on the expression level of the above-mentioned markergene in the control. For example, in certain embodiments, thepermissible range is taken as ±2S.D. based on the standard value. Oncethe standard value is determined, the testing method may be performed bymeasuring only the expression level in a biological sample from asubject and comparing the value with the determined standard value forthe control.

Expression levels of marker genes include transcription of the markergenes to mRNA, and translation into proteins. Therefore, one method oftesting for a cancer-related disease is performed based on a comparisonof the intensity of expression of mRNA corresponding to the markergenes, or the expression level of proteins encoded by the marker genes.

Probes

The measurement of the expression levels of marker genes in the testingfor a cancer-related disease can be carried out according to variousgene analysis methods. Specifically, one can use, for example, ahybridization technique using nucleic acids that hybridize to thesegenes as probes, or a gene amplification technique using DNA thathybridize to the marker genes as primers.

The probes or primers used for the testing can be designed based on thenucleotide sequences of the marker genes. The identification numbers forthe nucleotide sequences of the respective marker genes are describerherein.

Further, it is to be understood that genes of higher animals generallyaccompany polymorphism in a high frequency. There are also manymolecules that produce isoforms comprising mutually different amino acidsequences during the splicing process. Any gene associated with acancer-related disease that has an activity similar to that of a markergene is included in the marker genes, even if it has nucleotide sequencedifferences due to polymorphism or being an isoform.

It is also to be understood that the marker genes can include homologsof other species in addition to humans. Thus, unless otherwisespecified, the expression “marker gene” refers to a homolog of themarker gene unique to the species or a foreign marker gene which hasbeen introduced into an individual.

Also, it is to be understood that a “homolog of a marker gene” refers toa gene derived from a species other than a human, which can hybridize tothe human marker gene as a probe under stringent conditions. Suchstringent conditions are known to one skilled in the art who can selectan appropriate condition to produce an equal stringency experimentallyor empirically.

A polynucleotide comprising the nucleotide sequence of a marker gene ora nucleotide sequence that is complementary to the complementary strandof the nucleotide sequence of a marker gene and has at least 15nucleotides, can be used as a primer or probe. Thus, a “complementarystrand” means one strand of a double stranded DNA with respect to theother strand and which is composed of A:T (U for RNA) and G:C basepairs.

In addition, “complementary” means not only those that are completelycomplementary to a region of at least 15 continuous nucleotides, butalso those that have a nucleotide sequence homology of at least 40% incertain instances, 50% in certain instances, 60% in certain instances,70% in certain instances, at least 80%, 90%, and 95% or higher. Thedegree of homology between nucleotide sequences can be determined by analgorithm, BLAST, etc.

Such polynucleotides are useful as a probe to detect a marker gene, oras a primer to amplify a marker gene. When used as a primer, thepolynucleotide comprises usually 15 by to 100 bp, and in certainembodiments 15 by to 35 by of nucleotides. When used as a probe, a DNAcomprises the whole nucleotide sequence of the marker gene (or thecomplementary strand thereof), or a partial sequence thereof that has atleast 15 by nucleotides. When used as a primer, the 3′ region must becomplementary to the marker gene, while the 5′ region can be linked to arestriction enzyme-recognition sequence or a tag.

“Polynucleotides” may be either DNA or RNA. These polynucleotides may beeither synthetic or naturally-occurring. Also, DNA used as a probe forhybridization is usually labeled. Those skilled in the art readilyunderstand such labeling methods. Herein, the term “oligonucleotide”means a polynucleotide with a relatively low degree of polymerization.Oligonucleotides are included in polynucleotides.

Tests for Cancer-Related Diseases

Tests for a cancer-related disease using hybridization techniques can beperformed using, for example, Northern hybridization, dot blothybridization, or the DNA microarray technique. Furthermore, geneamplification techniques, such as the RT-PCR method may be used. Byusing the PCR amplification monitoring method during the geneamplification step in RT-PCR, one can achieve a more quantitativeanalysis of the expression of a marker gene.

In the PCR gene amplification monitoring method, the detection target(DNA or reverse transcript of RNA) is hybridized to probes that arelabeled with a fluorescent dye and a quencher which absorbs thefluorescence. When the PCR proceeds and Taq polymerase degrades theprobe with its 5′-3′ exonuclease activity, the fluorescent dye and thequencher draw away from each other and the fluorescence is detected. Thefluorescence is detected in real time. By simultaneously measuring astandard sample in which the copy number of a target is known, it ispossible to determine the copy number of the target in the subjectsample with the cycle number where PCR amplification is linear. Also,one skilled in the art recognizes that the PCR amplification monitoringmethod can be carried out using any suitable method.

The method of testing for a cancer-related disease can be also carriedout by detecting a protein encoded by a marker gene. Hereinafter, aprotein encoded by a marker gene is described as a “marker protein.” Forsuch test methods, for example, the Western blotting method, theimmunoprecipitation method, and the ELISA method may be employed usingan antibody that binds to each marker protein.

Antibodies used in the detection that bind to the marker protein may beproduced by any suitable technique. Also, in order to detect a markerprotein, such an antibody may be appropriately labeled. Alternatively,instead of labeling the antibody, a substance that specifically binds tothe antibody, for example, protein A or protein G, may be labeled todetect the marker protein indirectly. More specifically, such adetection method can include the ELISA method.

A protein or a partial peptide thereof used as an antigen may beobtained, for example, by inserting a marker gene or a portion thereofinto an expression vector, introducing the construct into an appropriatehost cell to produce a transformant, culturing the transformant toexpress the recombinant protein, and purifying the expressed recombinantprotein from the culture or the culture supernatant. Alternatively, theamino acid sequence encoded by a gene or an oligopeptide comprising aportion of the amino acid sequence encoded by a full-length cDNA arechemically synthesized to be used as an immunogen.

Furthermore, a test for a cancer-related disease can be performed usingas an index not only the expression level of a marker gene but also theactivity of a marker protein in a biological sample. Activity of amarker protein means the biological activity intrinsic to the protein.Various methods can be used for measuring the activity of each protein.

Even if a patient is not diagnosed as being affected with acancer-related disease in a routine test in spite of symptoms suggestingthese diseases, whether or not such a patient is suffering from acancer-related disease can be easily determined by performing a testaccording to the methods described herein.

More specifically, in certain embodiments, when the marker gene is oneof the genes described herein, an increase or decrease in the expressionlevel of the marker gene in a patient whose symptoms suggest at least asusceptibility to a cancer-related disease indicates that the symptomsare primarily caused by a cancer-related disease.

In addition, the tests are useful to determine whether a cancer-relateddisease is improving in a patient. In other words, the methods describedherein can be used to judge the therapeutic effect of a treatment for acancer-related disease. Furthermore, when the marker gene is one of thegenes described herein, an increase or decrease in the expression levelof the marker gene in a patient, who has been diagnosed as beingaffected by a cancer-related disease, implies that the disease hasprogressed more.

The severity and/or susceptibility to a cancer-related disease may alsobe determined based on the difference in expression levels. For example,when the marker gene is one of the genes described herein, the degree ofincrease in the expression level of the marker gene is correlated withthe presence and/or severity of a cancer-related disease.

Control of Expression of Marker

In addition, the expression itself of a marker gene can be controlled byintroducing a mutation(s) into the transcriptional regulatory region ofthe gene. Those skilled in the art understand such amino acidsubstitutions. Also, the number of amino acids that are mutated is notparticularly restricted, as long as the activity is maintained.Normally, it is within 50 amino acids, in certain non-limitingembodiments, within 30 amino acids, within 10 amino acids, or within 3amino acids. The site of mutation may be any site, as long as theactivity is maintained.

Screening Methods

In yet another aspect, there is provided herein screening methods forcandidate compounds for therapeutic agents to treat a cancer-relateddisease. One or more marker genes are selected from the group of genesdescribed herein. A therapeutic agent for a cancer-related disease canbe obtained by selecting a compound capable of increasing or decreasingthe expression level of the marker gene(s).

It is to be understood that the expression “a compound that increasesthe expression level of a gene” refers to a compound that promotes anyone of the steps of gene transcription, gene translation, or expressionof a protein activity. On the other hand, the expression “a compoundthat decreases the expression level of a gene”, as used herein, refersto a compound that inhibits any one of these steps.

In particular aspects, the method of screening for a therapeutic agentfor a cancer-related disease can be carried out either in vivo or invitro. This screening method can be performed, for example, by (1)administering a candidate compound to an animal subject; (2) measuringthe expression level of a marker gene(s) in a biological sample from theanimal subject; or (3) selecting a compound that increases or decreasesthe expression level of a marker gene(s) as compared to that in acontrol with which the candidate compound has not been contacted.

In still another aspect, there is provided herein a method to assess theefficacy of a candidate compound for a pharmaceutical agent on theexpression level of a marker gene(s) by contacting an animal subjectwith the candidate compound and monitoring the effect of the compound onthe expression level of the marker gene(s) in a biological samplederived from the animal subject. The variation in the expression levelof the marker gene(s) in a biological sample derived from the animalsubject can be monitored using the same technique as used in the testingmethod described above. Furthermore, based on the evaluation, acandidate compound for a pharmaceutical agent can be selected byscreening.

Kits

In another aspect, there is provided various diagnostic and test kits.In one embodiment, a kit is useful for assessing whether a patient isafflicted with a cancer-related disease. The kit comprises a reagent forassessing expression of a marker. In another embodiment, a kit is usefulfor assessing the suitability of a chemical or biologic agent forinhibiting a cancer-related disease in a patient. Such a kit comprises areagent for assessing expression of a marker, and may also comprise oneor more of such agents.

In a further embodiment, the kits are useful for assessing the presenceof cancer-related disease cells or treating cancer-related diseases.Such kits comprise an antibody, an antibody derivative or an antibodyfragment, which binds specifically with a marker protein or a fragmentof the protein. Such kits may also comprise a plurality of antibodies,antibody derivatives or antibody fragments wherein the plurality of suchantibody agents binds specifically with a marker protein or a fragmentof the protein.

In an additional embodiment, the kits are useful for assessing thepresence of cancer-related disease cells, wherein the kit comprises anucleic acid probe that binds specifically with a marker nucleic acid ora fragment of the nucleic acid. The kit may also comprise a plurality ofprobes, wherein each of the probes binds specifically with a markernucleic acid, or a fragment of the nucleic acid.

The compositions, kits and methods described herein can have thefollowing uses, among others: 1) assessing whether a patient isafflicted with a cancer-related disease; 2) assessing the stage of acancer-related disease in a human patient; 3) assessing the grade of acancer-related disease in a patient; 4) assessing the nature of acancer-related disease in a patient; 5) assessing the potential todevelop a cancer-related disease in a patient; 6) assessing thehistological type of cells associated with a cancer-related disease in apatient; 7) making antibodies, antibody fragments or antibodyderivatives that are useful for treating a cancer-related disease and/orassessing whether a patient is afflicted with a cancer-related disease;8) assessing the presence of cancer-related disease cells; 9) assessingthe efficacy of one or more test compounds for inhibiting acancer-related disease in a patient; 10) assessing the efficacy of atherapy for inhibiting a cancer-related disease in a patient; 11)monitoring the progression of a cancer-related disease in a patient; 12)selecting a composition or therapy for inhibiting a cancer-relateddisease in a patient; 13) treating a patient afflicted with acancer-related disease; 14) inhibiting a cancer-related disease in apatient; 15) assessing the harmful potential of a test compound; and 16)preventing the onset of a cancer-related disease in a patient at riskfor developing a cancer-related disease.

The kits are useful for assessing the presence of cancer-related diseasecells (e.g. in a sample such as a patient sample). The kit comprises aplurality of reagents, each of which is capable of binding specificallywith a marker nucleic acid or protein. Suitable reagents for bindingwith a marker protein include antibodies, antibody derivatives, antibodyfragments, and the like. Suitable reagents for binding with a markernucleic acid (e.g. a genomic DNA, an mRNA, a spliced mRNA, a cDNA, orthe like) include complementary nucleic acids. For example, the nucleicacid reagents may include oligonucleotides (labeled or non-labeled)fixed to a substrate, labeled oligonucleotides not bound with asubstrate, pairs of PCR primers, molecular beacon probes, and the like.

The kits may optionally comprise additional components useful forperforming the methods described herein. By way of example, the kit maycomprise fluids (e.g. SSC buffer) suitable for annealing complementarynucleic acids or for binding an antibody with a protein with which itspecifically binds, one or more sample compartments, an instructionalmaterial which describes performance of the method, a sample of normalcells, a sample of cancer-related disease cells, and the like.

Animal Model

Non-human animal model can be produced for assessment of at least onecancer-related disease. The method includes exposing the animal torepeated doses of at least one chemical believed to cause the cancer ifinterest. In certain aspects, the method further includes collecting oneor more selected samples from the animal; and comparing the collectedsample to one or more indicia of potential cancer initiation ordevelopment.

A method of producing the animal model includes: maintaining the animalin a specific chemical-free environment and sensitizing the animal withat least one chemical believed to cause the cancer. In certainembodiments, at least a part of the animal is sensitized by multiplesequential exposures.

A method of screening for an agent for effectiveness against at leastone cancer-related disease generally includes: administering at leastone agent to a test animal, determining whether the agent reduces oraggravates one or more symptoms of the cancer-related disease;correlating a reduction in one or more symptoms with effectiveness ofthe agent against the cancer-related disease; or correlating a lack ofreduction in one or more symptoms with ineffectiveness of the agent. Theanimal model is useful for assessing one or more metabolic pathways thatcontribute to at least one of initiation, progression, severity,pathology, aggressiveness, grade, activity, disability, mortality,morbidity, disease sub-classification or other underlying pathogenic orpathological feature of at least one cancer-related disease. Theanalysis can be by one or more of: hierarchical clustering, signaturenetwork construction, mass spectroscopy proteomic analysis, surfaceplasmon resonance, linear statistical modeling, partial least squaresdiscriminant analysis, and multiple linear regression analysis.

The animal model can be assessed for at least one cancer-relateddisease, by examining an expression level of one or more markers, or afunctional equivalent thereto.

The animal models can be used for the screening of therapeutic agentsuseful for treating or preventing a cancer-related disease. Accordingly,the methods are useful for identifying therapeutic agents for treatingor preventing a cancer-related disease. The methods compriseadministering a candidate agent to an animal model made by the methodsdescribed herein, assessing at least one cancer-related disease responsein the animal model as compared to a control animal model to which thecandidate agent has not been administered. If at least onecancer-related disease response is reduced in symptoms or delayed inonset, the candidate agent is an agent for treating or preventing thecancer-related disease.

The animal models for a cancer-related disease can include an animalwhere the expression level of one or more marker genes or a genefunctionally equivalent to the marker gene has been elevated in theanimal model. A “functionally equivalent gene” as used herein generallyis a gene that encodes a protein having an activity similar to a knownactivity of a protein encoded by the marker gene. A representativeexample of a functionally equivalent gene includes a counterpart of amarker gene of a subject animal, which is intrinsic to the animal.

The animal model for a cancer-related disease is useful for detectingphysiological changes due to a cancer-related disease. In certainembodiments, the animal model is useful to reveal additional functionsof marker genes and to evaluate drugs whose targets are the markergenes.

In one embodiment, an animal model for a cancer-related disease can becreated by controlling the expression level of a counterpart gene oradministering a counterpart gene. The method can include creating ananimal model for a cancer-related disease by controlling the expressionlevel of a gene selected from the group of genes described herein. Inanother embodiment, the method can include creating an animal model fora cancer-related disease by administering the protein encoded by a genedescribed herein, or administering an antibody against the protein. Itis to be also understood, that in certain other embodiments, the markercan be over-expressed such that the marker can then be measured usingappropriate methods.

In another embodiment, an animal model for a cancer-related disease canbe created by introducing a gene selected from such groups of genes, orby administering a protein encoded by such a gene.

In another embodiment, a cancer-related disease can be induced bysuppressing the expression of a gene selected from such groups of genesor the activity of a protein encoded by such a gene. An antisensenucleic acid, a ribozyme, or an RNAi can be used to suppress theexpression. The activity of a protein can be controlled effectively byadministering a substance that inhibits the activity, such as anantibody.

The animal model is useful to elucidate the mechanism underlying acancer-related disease and also to test the safety of compounds obtainedby screening. For example, when an animal model develops the symptoms ofa cancer-related disease, or when a measured value involved in a certaina cancer-related disease alters in the animal, a screening system can beconstructed to explore compounds having activity to alleviate thedisease.

As used herein, the expression “an increase in the expression level”refers to any one of the following: where a marker gene introduced as aforeign gene is expressed artificially; where the transcription of amarker gene intrinsic to the subject animal and the translation thereofinto the protein are enhanced; or where the hydrolysis of the protein,which is the translation product, is suppressed. As used herein, theexpression “a decrease in the expression level” refers to either thestate in which the transcription of a marker gene of the subject animaland the translation thereof into the protein are inhibited, or the statein which the hydrolysis of the protein, which is the translationproduct, is enhanced. The expression level of a gene can be determined,for example, by a difference in signal intensity on a DNA chip.Furthermore, the activity of the translation product—the protein—can bedetermined by comparing with that in the normal state.

It is also within the contemplated scope that the animal model caninclude transgenic animals, including, for example animals where amarker gene has been introduced and expressed artificially; marker geneknockout animals; and knock-in animals in which another gene has beensubstituted for a marker gene. A transgenic animal, into which anantisense nucleic acid of a marker gene, a ribozyme, a polynucleotidehaving an RNAi effect, or a DNA functioning as a decoy nucleic acid orsuch has been introduced, can be used as the transgenic animal. Suchtransgenic animals also include, for example, animals in which theactivity of a marker protein has been enhanced or suppressed byintroducing a mutation(s) into the coding region of the gene, or theamino acid sequence has been modified to become resistant or susceptibleto hydrolysis. Mutations in an amino acid sequence includesubstitutions, deletions, insertions, and additions.

THERAPEUTIC APPLICATIONS

The invention is widely applicable to a variety of situations where itis desirable to be able to regulate the level of gene expression, suchas by turning gene expression “on” and “off”, in a rapid, efficient andcontrolled manner without causing pleiotropic effects or cytotoxicity.The invention may be particularly useful for gene therapy purposes inhumans, in treatments for either genetic or acquired diseases. Thegeneral approach of gene therapy involves the introduction of one ormore nucleic acid molecules into cells such that one or more geneproducts encoded by the introduced genetic material are produced in thecells to restore or enhance a functional activity. For reviews on genetherapy approaches Anderson, et al. (1992; Miller et al. (1992);Friedmann et al. (1989); and Cournoyer et al. (1990). However, currentgene therapy vectors typically utilize constitutive regulatory elementswhich are responsive to endogenous transcriptions factors. These vectorsystems do not allow for the ability to modulate the level of geneexpression in a subject. In contrast, the regulatory system of theinvention provides this ability.

To use the system of the invention for gene therapy purposes, at leastone DNA molecule is introduced into cells of a subject in need of genetherapy (e.g., a human subject suffering from a genetic or acquireddisease) to modify the cells. The cells are modified to comprise: 1)nucleic acid encoding an inducible regulator of the invention in a formsuitable for expression of the inducible regulator in the host cells;and 2) an siRNA (e.g., for therapeutic purposes) operatively linked to atissue-specific promoter such as an s-ship1 promoter. A single DNAmolecule encoding components of the regulatory system of the inventioncan be used, or alternatively, separate DNA molecules encoding eachcomponent can be used. The cells of the subject can be modified ex vivoand then introduced into the subject or the cells can be directlymodified in vivo by conventional techniques for introducing nucleic acidinto cells. Thus, the regulatory system of the invention offers theadvantage over constitutive regulatory systems of allowing formodulation of the level of gene expression depending upon therequirements of the therapeutic situation.

Genes of particular interest to be knocked down or knocked out in cellsof a subject for treatment of genetic or acquired diseases include thoseencoding a deleterious gene product, such as an abnormal protein.Examples of non-limiting specific diseases include anemia, blood-relatedcancers, Parkinson's disease, and diabetes.

The present invention can be applied to develop autologous or allogeneiccell lines for therapeutical purposes. For example, gene therapyapplications of particular interest in cell and/or organ transplantationare utilized with the present invention. In exemplary embodiments,downregulation of transplantation antigens (such as, for example, bydownregulation of beta2-microglobulin expression via siRNA) allows fortransplantation of allogeneic cells while minimizing the risk ofrejection by the patient's immune system. The present invention wouldallow for a switch off of the RNAi in case of adverse effects (e.g.uncontrollable replication of the transplanted cells).

Cells types that can be subjected to the present invention includehematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, airwayepithelium, skin epithelium, islets, dopaminergic neurons,keratinocytes, and so forth. For further descriptions of cell types,genes and methods for gene therapy see e.g., Armentano et al. (1990);Wolff et al. (1990); Chowdhury et al. (1991); Ferry et al. (1991);Quantin et al (1992); Dai et al. (1992); van Beusechem et al. (1992);Rosenfeld et al. (1992); Kay et al. (1992); Cristiano et al (1993); Hwuet al (1993); and Herz and Gerard (1993).

In particular embodiments of the present invention, there is a method oftreating any disease condition amenable to treatment with an s-shippromoter. In specific embodiments, the method comprises preparing apolynucleotide construct having a region encoding a therapeutic ordiagnostic (marker) gene that is operably linked to a promoter, whereinthe gene encoded by the construct is for the treatment of the diseasecondition.

A. Pharmaceutical Formulations, Delivery, and Treatment Regimens

In an embodiment of the present invention, methods of treatment arecontemplated. An effective amount of the pharmaceutical composition,generally, is defined as that amount sufficient to detectably andrepeatedly to ameliorate, reduce, minimize or limit the extent of thedisease or its symptoms. More rigorous definitions may apply, includingelimination, eradication or cure of disease.

The routes of administration will vary, naturally, with the location andnature of the lesion, and include, e.g., intradermal, transdermal,parenteral, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral administration and formulation.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral and intraperitonealadministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

B. Combination Treatments

The compounds and methods of the present invention may be used in thecontext of traditional therapies. In order to increase the effectivenessof a treatment with the compositions of the present invention, it may bedesirable to combine these compositions with other agents effective inthe treatment of those diseases and conditions. For example, thetreatment of a cancer may be implemented with therapeutic compounds ofthe present invention and other anti-cancer therapies, such asanti-cancer agents or surgery. Likewise, the treatment of a vasculardisease or condition may involve both the present invention andconventional vascular agents or therapies.

Various combinations may be employed; for example, a host cell of thepresent invention is “A” and the secondary anti-cancer agent/therapy is“B”: TABLE-US-00005 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/BB/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/BB/A/A/A A/B/A/A A/A/B/A

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of that particular secondary therapy, taking into accountthe toxicity, if any, of the treatment. It is expected that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, as well as surgical intervention, maybe applied in combination with the described therapy.

All patents, patent applications and references cited herein areincorporated in their entirety by reference. While the invention hasbeen described and exemplified in sufficient detail for those skilled inthis art to make and use it, various alternatives, modifications andimprovements should be apparent without departing from the spirit andscope of the invention. One skilled in the art readily appreciates thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein.

The methods and reagents described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims. It will also be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modifications and variations of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

All of the compositions and/or methods and/or apparatus disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and/or apparatus and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (Glover ed.,1985); Oligonucleotide Synthesis (Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (Hames & Higgins eds.,1984); Transcription And Translation (Hames & Higgins eds., 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A PracticalGuide To Molecular Cloning (1984); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weirand Blackwell, eds., 1986); The Laboratory Rat, editor in chief: Mark A.Suckow; authors: Sharp and LaRegina. CRC Press, Boston, 1988, which areincorporated herein by reference) and chemical methods.

REFERENCES

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated be reference herein, and for convenience areprovided in the following bibliography.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

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REFERENCES FOR EXAMPLE II

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1-87. (canceled)
 88. A method to identify relative cancer risk in avertebrate, comprising identifying whether a test tissue sample of atest vertebrate comprises at least one transcribed ultra-conservedregions (T-UCR) expression profile that has a statistically significantcorrelation with at least one cancer, wherein a statisticallysignificant correlation indicates relative risk for a particular cancer.89. The method of claim 88, wherein the cancer is selected from thegroup consisting of: bladder cancer; esophageal cancer; lung cancer;stomach cancer; kidney cancer; cervical cancer; ovarian cancer; breastcancer; lymphoma; Ewing sarcoma; hematopoietic tumors; solid tumors;gastric cancer; colorectal cancer; brain cancer; epithelial cancer;nasopharyngeal cancer; uterine cancer; hepatic cancer; head-and-neckcancer; renal cancer; male germ cell tumors; malignant mesothelioma;myelodysplastic syndrome; pancreatic or biliary cancer; prostate cancer;thyroid cancer; urothelial cancer; renal cancer; Wilm's tumor; smallcell lung cancer; melanoma; skin cancer; osteosarcoma; neuroblastoma;leukemia (acute lymphocytic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia); glioblastoma multiforme; medulloblastoma;lymphoplasmacytoid lymphoma; and rhabdomyosarcoma.
 90. A method of claim88, wherein the at least one T-UCR are the seven T-UCRs of uc.347through uc.353.
 91. A method claim 88, wherein the at least one T-UCRare the two T-UCRs of uc.349A(P) and uc.352(N).
 92. A method of claim91, wherein a statistically significant correlation indicates malignantB-CLL CD5 positive cells.
 93. A method of claim 88, wherein the at leastone T-UCR are the five UCRs of uc.269A(N), uc.160(N), uc.215(N),uc.346A(P) and uc.348(N).
 94. A method of claim 93, wherein astatistically significant correlation indicates CLL risk.
 95. A methodof claim 88, wherein the at least one T-UCR is uc.73(P).
 96. A method ofclaim 88, wherein a statistically significant correlation indicatescolon cancer risk.
 97. A method to identify molecules useful to affectexpression of a T-UCR associated with increased cancer risk, comprisingintroducing a test molecule to a T-UCR microarray and identifying thosemolecules which affect expression.
 98. The method of claim 97, whereinthe cancer is selected from the group consisting of: bladder cancer;esophageal cancer; lung cancer; stomach cancer; kidney cancer; cervicalcancer; ovarian cancer; breast cancer; lymphoma; Ewing sarcoma;hematopoietic tumors; solid tumors; gastric cancer; colorectal cancer;brain cancer; epithelial cancer; nasopharyngeal cancer; uterine cancer;hepatic cancer; head-and-neck cancer; renal cancer; male germ celltumors; malignant mesothelioma; myelodysplastic syndrome; pancreatic orbiliary cancer; prostate cancer; thyroid cancer; urothelial cancer;renal cancer; Wilm's tumor; small cell lung cancer; melanoma; skincancer; osteosarcoma; neuroblastoma; leukemia (acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia);glioblastoma multiforme; medulloblastoma; lymphoplasmacytoid lymphoma;and rhabdomyosarcoma.
 99. A composition of matter, comprising aT-UCR/miRNA interacting pair selected from the group consistingessentially of: uc.160:: miR-24; uc.160:: miR-155; uc.160:: miR-223;uc.160::miR-146a; uc.346A::miR-155; and uc.-348::miR-29b.
 100. A cDNAthat is complementary to a T-UCR selected from the group consistingessentially of: uc.347; uc.348; uc.349; uc.350; uc.351; uc352; uc.353;uc.269A(N), uc.160(N), uc.215(N), uc.346A(P); uc.348(N); and uc.73(P).101. A cDNA corresponding to uc.246(E).
 102. The cDNA of claim 101cloned and expressed by standard methods.
 103. A cDNA corresponding touc.269A(N).
 104. The cDNA of claim 103 cloned and expressed by standardmethods.
 105. A method for assessing the prognosis of a cancer patientcomprising correlating expression of UCRs and miRNAs in test sample froma cancer patient and identifying those correlations which arestatistically significant so as to assess the prognosis.
 106. The methodof claim 105, wherein the cancer is selected from the group consistingof: bladder cancer; esophageal cancer; lung cancer; stomach cancer;kidney cancer; cervical cancer; ovarian cancer; breast cancer; lymphoma;Ewing sarcoma; hematopoietic tumors; solid tumors; gastric cancer;colorectal cancer; brain cancer; epithelial cancer; nasopharyngealcancer; uterine cancer; hepatic cancer; head-and-neck cancer; renalcancer; male germ cell tumors; malignant mesothelioma; myelodysplasticsyndrome; pancreatic or biliary cancer; prostate cancer; thyroid cancer;urothelial cancer; renal cancer; Wilm's tumor; small cell lung cancer;melanoma; skin cancer; osteosarcoma; neuroblastoma; leukemia (acutelymphocytic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia); glioblastoma multiforme; medulloblastoma; lymphoplasmacytoidlymphoma; and rhabdomyosarcoma.
 107. The method of claim 105, whereinthe cancer is CLL.
 108. The method of claim 105, wherein the correlationcomprises the identification of miRNA::T-UCR interaction.
 109. A methodfor assessing the prognosis of a cancer patient comprising identifyingany altered genomic nc-UCGs in test sample from a cancer patient so asto assess the prognosis.
 110. The method of claim 109, wherein thepatient has a cancer is selected from the group consisting of: bladdercancer; esophageal cancer; lung cancer; stomach cancer; kidney cancer;cervical cancer; ovarian cancer; breast cancer; lymphoma; Ewing sarcoma;hematopoietic tumors; solid tumors; gastric cancer; colorectal cancer;brain cancer; epithelial cancer; nasopharyngeal cancer; uterine cancer;hepatic cancer; head-and-neck cancer; renal cancer; male germ celltumors; malignant mesothelioma; myelodysplastic syndrome; pancreatic orbiliary cancer; prostate cancer; thyroid cancer; urothelial cancer;renal cancer; Wilm's tumor; small cell lung cancer; melanoma; skincancer; osteosarcoma; neuroblastoma; leukemia (acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia);glioblastoma multiforme; medulloblastoma; lymphoplasmacytoid lymphoma;and rhabdomyosarcoma.
 111. A method of claim 109, wherein the cancerpatient has leukemia.
 112. A method of diagnosing whether a subject has,or is at risk for developing, a cancer linked to a cancer-associatedchromosomal feature, comprising evaluating the status in the subject ofat least one UCR gene located in close proximity to thecancer-associated chromosomal feature, by measuring in a test samplefrom said subject the level of at least one UCR gene product from theUCR gene in the test sample, wherein an alteration in the level of UCRgene product in the test sample relative to the level of correspondingUCR gene product in a control sample is indicative of the subject eitherhaving, or being at risk for developing, the cancer.
 113. A method ofclaim 112, wherein the UCR gene is selected from the group consistingof: a cluster of seven UCRs of uc.347 through uc.353; and combinationsthereof.
 114. A method of claim 112, wherein the cancer is a leukemia.115. The method of claim 112, wherein the cancer-associated chromosomalfeature is selected from the group consisting of: a cancer-associatedgenomic region; a chromosomal fragile site; a human papillomavirusintegration site; and a homeobox gene or gene cluster.
 116. The methodof claim 112, wherein the cancer is selected from the group consistingof: bladder cancer; esophageal cancer; lung cancer; stomach cancer;kidney cancer; cervical cancer; ovarian cancer; breast cancer; lymphoma;Ewing sarcoma; hematopoietic tumors; solid tumors; gastric cancer;colorectal cancer; brain cancer; epithelial cancer; nasopharyngealcancer; uterine cancer; hepatic cancer; head-and-neck cancer; renalcancer; male germ cell tumors; malignant mesothelioma; myelodysplasticsyndrome; pancreatic or biliary cancer; prostate cancer; thyroid cancer;urothelial cancer; renal cancer; Wilm's tumor; small cell lung cancer;melanoma; skin cancer; osteosarcoma; neuroblastoma; leukemia (acutelymphocytic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia); glioblastoma multiforme; medulloblastoma; lymphoplasmacytoidlymphoma; and rhabdomyosarcoma.
 117. A method of diagnosing whether asubject has, or is at risk for developing, a cancer linked to acancer-associated chromosomal feature, comprising evaluating the statusin the subject of at least one UCR gene located in close proximity tothe cancer-associated chromosomal feature, by analyzing the at least oneUCR gene in the test sample for a deletion, mutation or amplification,wherein detection of a deletion, mutation and/or amplification in theUCR gene as compared to the corresponding UCR gene in the control sampleis indicative of the subject either having, or being at risk fordeveloping, the cancer.
 118. A method of claim 117, wherein the UCR geneis selected from the group consisting of: a cluster of seven UCRs ofuc.347 through uc.353; and combinations thereof.
 119. A method of claim117, wherein the cancer is a leukemia.
 120. The method of claim 117,wherein the cancer-associated chromosomal feature is selected from thegroup consisting of: a cancer-associated genomic region; a chromosomalfragile site; a human papillomavirus integration site; and a homeoboxgene or gene cluster.
 121. The method of claim 117, wherein the canceris selected from the group consisting of: bladder cancer; esophagealcancer; lung cancer; stomach cancer; kidney cancer; cervical cancer;ovarian cancer; breast cancer; lymphoma; Ewing sarcoma; hematopoietictumors; solid tumors; gastric cancer; colorectal cancer; brain cancer;epithelial cancer; nasopharyngeal cancer; uterine cancer; hepaticcancer; head-and-neck cancer; renal cancer; male germ cell tumors;malignant mesothelioma; myelodysplastic syndrome; pancreatic or biliarycancer; prostate cancer; thyroid cancer; urothelial cancer; renalcancer; Wilm's tumor; small cell lung cancer; melanoma; skin cancer;osteosarcoma; neuroblastoma; leukemia (acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia); glioblastomamultiforme; medulloblastoma; lymphoplasmacytoid lymphoma; andrhabdomyosarcoma.
 122. A method of diagnosing whether a subject has, oris at risk for developing, a cancer linked to a cancer-associatedchromosomal feature, comprising evaluating the status in the subject ofat least one UCR gene located in close proximity to thecancer-associated chromosomal feature, by measuring the copy number ofthe at least one UCR gene in the test sample, wherein a copy numberother than two for an UCR gene on a somatic chromosome or sex chromosomein a female, or other than one for an UCR gene on a sex chromosome in amale, is indicative of the subject either having, or being at risk fordeveloping, the cancer.
 123. The method of claim 122, wherein thecancer-associated chromosomal feature is selected from the groupconsisting of: a cancer-associated genomic region; a chromosomal fragilesite; a human papillomavirus integration site; and a homeobox gene orgene cluster.
 124. The method of claim 122, wherein the cancer isselected from the group consisting of: bladder cancer; esophagealcancer; lung cancer; stomach cancer; kidney cancer; cervical cancer;ovarian cancer; breast cancer; lymphoma; Ewing sarcoma; hematopoietictumors; solid tumors; gastric cancer; colorectal cancer; brain cancer;epithelial cancer; nasopharyngeal cancer; uterine cancer; hepaticcancer; head-and-neck cancer; renal cancer; male germ cell tumors;malignant mesothelioma; myelodysplastic syndrome; pancreatic or biliarycancer; prostate cancer; thyroid cancer; urothelial cancer; renalcancer; Wilm's tumor; small cell lung cancer; melanoma; skin cancer;osteosarcoma; neuroblastoma; leukemia (acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia); glioblastomamultiforme; medulloblastoma; lymphoplasmacytoid lymphoma; andrhabdomyosarcoma.
 125. A method of claim 122, wherein the UCR gene isselected from the group consisting of: a cluster of seven UCRs of uc.347through uc.353; and combinations thereof.
 126. A method of claim 122,wherein the cancer is a leukemia.
 127. A method of diagnosing whether asubject has, or is at risk for developing, a cancer, comprising: (1)reverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides; (2) hybridizing thetarget oligodeoxynucleotides to a microarray comprising one or more UCRspecific probe oligonucleotides to provide a hybridization profile forthe test sample; and (3) comparing the test sample hybridization profileto a hybridization profile generated from a control sample, wherein analteration in the signal is indicative of the subject either having, orbeing at risk for developing, the cancer.
 128. The method of claim 127,wherein the cancer is selected from the group consisting of: bladdercancer; esophageal cancer; lung cancer; stomach cancer; kidney cancer;cervical cancer; ovarian cancer; breast cancer; lymphoma; Ewing sarcoma;hematopoietic tumors; solid tumors; gastric cancer; colorectal cancer;brain cancer; epithelial cancer; nasopharyngeal cancer; uterine cancer;hepatic cancer; head-and-neck cancer; renal cancer; male germ celltumors; malignant mesothelioma; myelodysplastic syndrome; pancreatic orbiliary cancer; prostate cancer; thyroid cancer; urothelial cancer;renal cancer; Wilm's tumor; small cell lung cancer; melanoma; skincancer; osteosarcoma; neuroblastoma; leukemia (acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia);glioblastoma multiforme; medulloblastoma; lymphoplasmacytoid lymphoma;and rhabdomyosarcoma.
 129. The method of claim 127, wherein the canceris B-cell chronic lymphocytic leukemia.
 130. A method of diagnosingwhether a subject has, or is at risk for developing, a cancer associatedwith one or more adverse prognostic markers in a subject, comprising:(1) reverse transcribing RNA from a test sample obtained from thesubject to provide a set of target oligodeoxynucleotides; (2)hybridizing the target oligodeoxynucleotides to a microarray comprisingone or more UCR-specific probe oligonucleotides to provide ahybridization profile for said test sample; and (3) comparing the testsample hybridization profile to a hybridization profile generated from acontrol sample, wherein an alteration in the signal is indicative of thesubject either having, or being at risk for developing, the cancer. 131.The method of claim 130, wherein the cancer is selected from the groupconsisting of: bladder cancer; esophageal cancer; lung cancer; stomachcancer; kidney cancer; cervical cancer; ovarian cancer; breast cancer;lymphoma; Ewing sarcoma; hematopoietic tumors; solid tumors; gastriccancer; colorectal cancer; brain cancer; epithelial cancer;nasopharyngeal cancer; uterine cancer; hepatic cancer; head-and-neckcancer; renal cancer; male germ cell tumors; malignant mesothelioma;myelodysplastic syndrome; pancreatic or biliary cancer; prostate cancer;thyroid cancer; urothelial cancer; renal cancer; Wilm's tumor; smallcell lung cancer; melanoma; skin cancer; osteosarcoma; neuroblastoma;leukemia (acute lymphocytic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia); glioblastoma multiforme; medulloblastoma;lymphoplasmacytoid lymphoma; and rhabdomyosarcoma.
 132. The method ofclaim 130, wherein the cancer is B-cell chronic lymphocytic leukemia.133. A method of diagnosing whether a subject has, or is at risk fordeveloping, a cancer, comprising analyzing in a test sample from saidsubject at least one UCR gene or gene product associated with a cancer,wherein detection of a mutation in the UCR gene or gene product, ascompared to the corresponding UCR gene or gene product in a controlsample, is indicative of the subject having, or being at risk fordeveloping, the cancer.
 134. The method of claim 133, wherein the canceris selected from the group consisting of: bladder cancer; esophagealcancer; lung cancer; stomach cancer; kidney cancer; cervical cancer;ovarian cancer; breast cancer; lymphoma; Ewing sarcoma; hematopoietictumors; solid tumors; gastric cancer; colorectal cancer; brain cancer;epithelial cancer; nasopharyngeal cancer; uterine cancer; hepaticcancer; head-and-neck cancer; renal cancer; male germ cell tumors;malignant mesothelioma; myelodysplastic syndrome; pancreatic or biliarycancer; prostate cancer; thyroid cancer; urothelial cancer; renalcancer; Wilm's tumor; small cell lung cancer; melanoma; skin cancer;osteosarcoma; neuroblastoma; leukemia (acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia); glioblastomamultiforme; medulloblastoma; lymphoplasmacytoid lymphoma; andrhabdomyosarcoma.
 135. The method of claim 133, wherein the at least oneUCR gene or gene product is selected from the group consisting of: acluster of seven UCRs of uc.347 through uc.353, and combinationsthereof.
 136. The method of claim 133, wherein the cancer is B-cellchronic lymphocytic leukemia.
 137. A pharmaceutical compositioncomprising a UCR gene product and a pharmaceutically-acceptable carrier,wherein the isolated UCR gene product is from a UCR gene located inclose proximity to a cancer-associated chromosomal feature.
 138. Thepharmaceutical composition of claim 137, wherein the cancer-associatedchromosomal feature is selected from the group consisting of acancer-associated genomic region, and a chromosomal fragile site.
 139. Apharmaceutical composition comprising a nucleic acid encoding anisolated UCR gene product from a UC gene located in close proximity to acancer-associated chromosomal feature, and a pharmaceutically-acceptablecarrier.
 140. The pharmaceutical composition of claim 139, wherein thecancer-associated chromosomal feature is selected from the groupconsisting of a cancer-associated genomic region, a chromosomal fragilesite, a human papillomavirus integration site, and a homeobox gene orgene cluster.
 141. A method of treating cancer in a subject, comprising:(1) providing a subject who has a cancer associated with acancer-associated chromosomal feature, in which at least one isolatedUCR gene product from a UCR gene located in close proximity to thecancer-associated chromosomal feature is down-regulated or up-regulatedin cancer cells of the subject as compared to control cells; and (2) (a)when the at least one isolated miR gene product is down-regulated in thecancer cells, administering to the subject an effective amount of atleast one isolated miR gene product from the at least one UCR gene, suchthat proliferation of cancer cells in the subject is inhibited; or (b)when the at least one isolated UCR gene product is up-regulated in thecancer cells, administering to the subject an effective amount of atleast one compound for inhibiting expression of the at least one UCRgene, such that proliferation of cancer cells in the subject isinhibited.
 142. The method of claim 141, wherein the cancer-associatedchromosomal feature is selected from the group consisting of acancer-associated genomic region, and a chromosomal fragile site. 143.The method of treating cancer associated with a cancer-associatedchromosomal feature, comprising: (1) determining the amount of UC geneproduct expressed from at least one UCR gene located in close proximityto the cancer-associated chromosomal feature in cancer cells from asubject, relative to control cells; and (2) altering the amount of UCgene product expressed in the cancer cells by: (i) administering to thesubject an effective amount of at least one isolated miR gene productfrom the UCR gene, if the amount of the UCR gene product expressed inthe cancer cells is less than amount of the miR gene product expressedin control cells; or (ii) administering to the subject an effectiveamount of at least one compound for inhibiting expression of the atleast one UCR gene, if the amount of the UCR gene product expressed inthe cancer cells is greater than the amount of the UCR gene productexpressed in control cells, such that proliferation of cancer cells inthe subject is inhibited.
 144. The method of claim 143, wherein thecancer-associated chromosomal feature is selected from the groupconsisting of a cancer-associated genomic region, and a chromosomalfragile site.
 145. The method of claim 143, wherein thecancer-associated chromosomal feature is a chromosomal fragile site.146. The method of claim 143, wherein the cancer-associated chromosomalfeature is a cancer-associated genomic region.
 147. A composition ofmatter comprising an isolated T-UCR associated with cancer, wherein saidassociation is statistically significant, and the composition is amarker for assessing one or more metabolic pathways that contribute toat least one of initiation, progression, severity, pathology,aggressiveness, grade, activity, disability, mortality, morbidity,disease sub-classification or other underlying pathogenic orpathological feature of at least one lung cancer-related disease.
 148. Amethod of identifying a potential for the initiation or development ofat least one cancer-related disease in a subject, comprising identifyingthe presence of a composition of claim 147 in a test sample.
 149. Amethod of claim 148, wherein all method steps are performed in vitro.150. A reagent for testing for a cancer-related disease, wherein thereagent comprises a polynucleotide comprising the nucleotide sequence ofa composition of claim 147 or a nucleotide sequence complementary to thenucleotide sequence of the marker.
 151. A reagent for testing for acancer-related disease, wherein the reagent comprises an antibody thatrecognizes a composition of claim
 147. 152. A DNA chip for testing for acancer-related disease, on which a probe has been immobilized to assay acomposition of claim
 147. 153. A method of assessing the effectivenessof a therapy to prevent, diagnose and/or treat at least one lungcancer-related disease comprising: (1) subjecting an animal to a therapywhose effectiveness is being assessed, and (2) determining the level ofeffectiveness of the treatment being tested in treating or preventingthe lung cancer-related disease by evaluating a composition of claim147.
 154. A method of claim 153, wherein the candidate therapeutic agentcomprises one or more of: pharmaceutical compositions, nutraceuticalcompositions, and homeopathic compositions.
 155. A method of claim 153,wherein the therapy being assessed is for use in a human subject. 156.The method of claim 153, wherein the method is not a method of treatmentof the human or animal body by surgery or therapy.
 157. A method ofassessing the potential of at least one material for an ability toinitiate a cancer-related disease response in an animal model, themethod providing: (1) measuring one or more of up- or down-regulatedmarkers of a composition of claim 60 after exposure of the animal to oneor more materials in amounts sufficient to initiate a cancer-relateddisease response in the animal; and (2) determining whether at least oneof the up- or down-regulated markers has the ability to initiate acancer-related disease response.
 158. A composition of matter comprisingan isolated T-UCR transcript associated with cancer, wherein saidassociation is statistically significant, and the composition is amarker for assessing one or more metabolic pathways that contribute toat least one of initiation, progression, severity, pathology,aggressiveness, grade, activity, disability, mortality, morbidity,disease sub-classification or other underlying pathogenic orpathological feature of at least one lung cancer-related disease.
 159. Amethod of identifying a potential for the initiation or development ofat least one cancer-related disease in a subject, comprising identifyingthe presence of a composition of claim 158 in a test sample.
 160. Amethod of claim 158, wherein all method steps are performed in vitro.161. A reagent for testing for a cancer-related disease, wherein thereagent comprises a polynucleotide comprising the nucleotide sequence ofa composition of claim 158 or a nucleotide sequence complementary to thenucleotide sequence of the marker.
 162. A reagent for testing for acancer-related disease, wherein the reagent comprises an antibody thatrecognizes a composition of claim
 158. 163. A DNA chip for testing for acancer-related disease, on which a probe has been immobilized to assay acomposition of claim
 158. 164. A method of assessing the effectivenessof a therapy to prevent, diagnose and/or treat at least one lungcancer-related disease comprising: (1) subjecting an animal to a therapywhose effectiveness is being assessed, and (2) determining the level ofeffectiveness of the treatment being tested in treating or preventingthe lung cancer-related disease by evaluating a composition of claim158.
 165. A method of claim 164, wherein the candidate therapeutic agentcomprises one or more of: pharmaceutical compositions, nutraceuticalcompositions, and homeopathic compositions.
 166. A method of claim 164,wherein the therapy being assessed is for use in a human subject. 167.The method of claim 164, wherein the method is not a method of treatmentof the human or animal body by surgery or therapy.
 168. A pharmaceuticalcomposition for treating a cancer-related disease, comprising: at leastone UCR gene product selected from the group consisting of a cluster ofseven UCRs of uc.347 through uc.353; and combinations thereof; and, apharmaceutically-acceptable carrier.
 169. The pharmaceutical compositionof claim 168, wherein the UCR gene product that is up- or down-regulatedin cancer cells is relative to suitable control cells.
 170. Thepharmaceutical composition of claim 168, wherein the cancer-relateddisease is adenocarcinoma.
 171. A pharmaceutical composition fortreating a lung cancer, comprising at least one UCRexpression-inhibition compound, and a pharmaceutically-acceptablecarrier, wherein the at least one UCR expression-inhibition compound isspecific for a UCR gene product selected from the group consisting of acluster of seven UCRs of uc.347 through uc.353; and combinationsthereof.
 172. The pharmaceutical composition of claim 171, wherein theat least one UCR expression-inhibition compound is specific for a UCRgene product that is up- or down-regulated in cancer cells relative tosuitable control cells.
 173. A kit for screening for a candidatecompound for a therapeutic agent to treat a cancer-related disease,wherein the kit comprises: a reagent capable of identifying acomposition of claim
 147. 174. The kit of claim 173, wherein reagent isan antibody or an antibody fragment.
 175. The kit of claim 173, whereinthe reagent is labeled, radio-labeled, or biotin-labeled, and/or whereinthe antibody or antibody fragment is radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled.
 176. The kit of claim 173,wherein the reagent comprises one or more of: an antibody, a probe towhich the reagent is attached or is attachable, and an immobilized metalchelate.
 177. A kit for screening for a candidate compound for atherapeutic agent to treat a cancer-related disease, wherein the kitcomprises: a reagent capable of identifying a composition of claim 158.178. The kit of claim 177, wherein reagent is an antibody or an antibodyfragment.
 179. The kit of claim 177, wherein the reagent is labeled,radio-labeled, or biotin-labeled, and/or wherein the antibody orantibody fragment is radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled.
 180. The kit of claim 177,wherein the reagent comprises one or more of: an antibody, a probe towhich the reagent is attached or is attachable, and an immobilized metalchelate.
 181. A screening test for agents useful in a lungcancer-related disease research or treatment, comprising: contacting acomposition of claim 147 with a test agent, and determining whether thetest agent modulates the activity of the composition.
 182. A screeningtest of claim 181, wherein all method steps are performed in vitro. 183.A test of claim 181, wherein determination of modulation is assessed bydetecting the presence of a transcribed polynucleotide or portionthereof, wherein the transcribed polynucleotide comprises a codingregion of the composition.
 184. A test of claim 181, wherein contactingoccurs via a sample of a cancer-associated body fluid or tissue.
 185. Atest of claim 181, wherein the sample comprises cells obtained from thepatient.
 186. A screening test for agents useful in a lungcancer-related disease research or treatment, comprising: contacting acomposition of claim 158 with a test agent, and determining whether thetest agent modulates the activity of the composition.
 187. A screeningtest of claim 186, wherein all method steps are performed in vitro. 188.A test of claim 186, wherein determination of modulation is assessed bydetecting the presence of a transcribed polynucleotide or portionthereof, wherein the transcribed polynucleotide comprises a codingregion of the composition.
 189. A test of claim 186, wherein contactingoccurs via a sample of a cancer-associated body fluid or tissue.
 190. Amethod of claim 186, wherein the sample comprises cells obtained fromthe patient.
 191. A microarray for predicting the presence of acancer-related disease in a subject comprising an antibody directed to acomposition of claim
 147. 192. A microarray for predicting the presenceof a cancer-related disease in a subject comprising an antibody directedto a composition of claim
 158. 193. A method for treating, preventing,reversing or limiting the severity of a cancer-related diseasecomplication in an individual in need thereof, comprising: administeringto the individual an agent that interferes with at least onecancer-related disease response signaling pathway, in an amountsufficient to interfere with such signaling, wherein the agent comprisesat least one UCR gene product.
 194. A method to interfere with at leaston lung cancer-related disease response signaling pathway, for themanufacture of a medicament for treating, preventing, reversing orlimiting the severity of a cancer-related disease complication in anindividual, wherein the agent comprises at least one UCR gene product.195. A method of treating, preventing, reversing or limiting theseverity of a cancer-related disease complication in an individual inneed thereof, comprising administering to the individual an agent thatinterferes with at least one cancer-related disease response cascade,wherein the agent comprises at least one UCR gene product.
 196. A methodto interfere with at least one cancer-related disease response cascade,for the manufacture of a medicament for treating, preventing, reversingor limiting the severity of a cancer-related disease complication in anindividual, wherein the agent comprises at least one UCR gene product.197. A computer-readable medium comprising a database having a pluralityof digitally-encoded reference profiles, wherein at least a firstreference profile represents a level of at least a first marker in oneor more samples from one or more subjects exhibiting an indicia of acancer-related disease response, wherein the marker comprises one ormore UCR gene products.
 198. A computer readable medium of claim 197,including at least a second reference profile that represents a level ofat least a second marker in one or more samples from one or moresubjects exhibiting indicia of a cancer-related disease response; orsubjects having a cancer-related disease.
 199. A computer system fordetermining whether a subject has, is predisposed to having, or has apoor survival prognosis for, a cancer-related disease, comprising thedatabase of claim 198, and a server comprising a computer-executablecode for causing the computer to receive a profile of a subject,identify from the database a matching reference profile that isdiagnostically relevant to the subject profile, and generate anindication of whether the subject has, or is predisposed to having, acancer-related disease.
 200. A computer-assisted method for evaluatingthe presence, absence, nature or extent of a cancer-related disease in asubject, comprising: (1) providing a computer comprising a model oralgorithm for classifying data from a sample obtained from the subject,wherein the classification includes analyzing the data for the presence,absence or amount of at least one marker, and wherein the markercomprises one or more UCR genes or gene products, provided that the UCRgene or gene product has been shown to be statistically significant insuch association with cancer-related disease; (2) inputting data fromthe biological sample obtained from the subject; and, (3) classifyingthe biological sample to indicate the presence, absence, nature orextent of a cancer-related disease.
 201. The method of claim 200,wherein the at least one UCR gene product and combinations thereofincludes isolated variants or biologically-active fragments orfunctional equivalents thereof, or antibodies that bind thereto.
 202. Amethod of claim 200, wherein the UCR gene product is selected from thegroup consisting of a cluster of seven UCRs of uc.347 through uc.353;and combinations thereof.
 203. An animal model for cancer wherein atleast one of an altered expression of one or more UCR gene products ispresent.
 204. An animal model of claim 203, wherein the animal model isa nonhuman vertebrate.
 205. An animal model of claim 203, wherein theanimal model is a mouse, rat, rabbit, or primate.